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https://openalex.org/W1990077284	https://ahea.pitt.edu/ojs/index.php/ahea/article/download/16/232	English	null	"A megcsalt férj", or Cunningly Lingual Wives in Hungarian Ballad Tradition	Hungarian cultural studies	2,009	cc-by	4,971	Abstract Abstract The European ballad, an orally-performed narrative song, developed in the medieval period with many cross-fertilizations among ballad types in various language areas. Nevertheless, to date there have appeared only a handful of comparative studies of these pan-european themes, with investigations dominated by the Finnish geographical school, whose primary interest is in finding genetic archetypes. In this study, my aim is, rather, to do a typological and stylistic analysis of one wide-circulating song-type, known in many variants throughout the continent, some in comic and others in tragic versions. The ballad I shall analyze appears in Hungarian in several variants as "A Megcsalt ferj," in Anglo-American tradition -- recorded in over 400 variants – the ballad is known as "Our Goodman," or "The Cuckold's Song," or, in more blatantly obscene versions as "The Old Man Came Home" and "Home Drunk Cam' I". There also exist Spanish, Catalan, French, Italian, and even Yiddish versions, all of which I shall be taking into consideration In this paper I shall study one widely circulating European ballad tradition concerning wifely infidelity to illustrate that, although details among versions are temporally and culturally variable, they all belong to one narrative deep structure having to do with “cunningly lingual” wives and cuckolded husband. These adulterous wives are a subcategory of the persistent antifeminist stereotype of the “unruly woman”, whose too active mouth, whether its voraciousness, garrulousness, or verbal cunning, implies bodily misrule, a topsy-turvy displacement of her even more fearful orifice, the vagina. I can only mention noteworthy highlights from several traditions of ballads, so as to be able to concentrate on the Hungarian corpus. Through a gender-conscious reading I will show that these retold tales that pretend to be about conjugal relations are merely another variant of misogynist male discourse on women, where the “misogyny reveals far more about masculinity and male views of the feminine than about real women” (Gaunt 71). In addition, hegemonic masculinist readings, including by scholars, have been complicit in a directed reading away from the misogyny. The earliest known variants of the ballad I shall discuss survive in two traditional medieval Spanish ballads, known also in hundreds of modern oral variants. Beyond Hispanic tradition, versions also appears in many other languages, including French, Italian, in the Balkans, and even in Yiddish, with the most widespread variants are in Anglo-American tradition (for a detailed analysis of these traditions see Vasvari 2008). 1 1 1 “A Megcsalt férj”, or Cunningly Lingual Wives in Hungarian Ballad Tradition Louise O. Vasvári (Stony Brook University) Abstract All these ballads narrate how a husband returns home unexpectedly and surprises his wife with her lover. The lover’s presence is gradually revealed through a series of tell-tale clues – typically a horse, sword, a hat, and perhaps physical evidence, such as his clothes, or, finally, even the sight of his moustache in the bed. The narrative core of the ballad is a reiterative “testing dialogue,“ in which the husband questions his wife about all the signs of her infidelity and through a smokescreen of cunning semiotic ruses she attempts to manipulate all the signs of her guilt by relocating them in a new context created by her women’s speech. She might claim, for example, that the saddled horse standing outside is a gift from her father for the husband, or, more ludicrously, that it is 2 the cow that the maid left there, that the lover’s blue dolman on the bed is actually a mildewed bedspread, or, in an obscene variant, even that the lover’s genitalia are parsnips, or perhaps beets! While the structure (but not the details) of the dramatic testing dialogue occur in almost all versions of the ballad, there are a variety of possible endings, ranging from the sadistic to the merely punitive, to the ambiguous and the comic, with the latter ending with the wife’s linguistic and sexual victory -- a carnivalesque temporary suspension or reversal of normal rules of patriarchal culture. It is the Hispanic variants of The Adulterous Wife, that have been most widely studied, with some earlier critics claiming that since the common origin of the plot is from a medieval comic fabliau, the originality of the Spanish version resides in having given the theme a morally superior tragic orientation, where the wife gets her deserved punishment. In fact, however, although the two extant oldest versions end with the wife’s death, among the hundreds of modern versions there are also many humorous ones. And, as we shall see, Hungarian tradition contains a far more violently sadistic versions than any known in earlier Hispanic tradition. Although ballad collection in Hungary started very late, with the first printed collections dating, not coincidentally, from the period of the war of independence in 1846-48, it is likely that some of the earliest themes (such as that of the building sacrifice) go back to ancient traditions (Balassa & Ortutay: 515, Dundes). Abstract 3 3 Losing patience, the husband finally breaks down the door and demands the key to the chest in which the lover is hiding. The wife claims to have lost the key in the garden but he kicks the chest open, finds the lover and cuts off his head. The scene is familiar from fabliau tradition, with the sexual suggestiveness of phallic key, a folk symbol for the male organ (as, for example, in the jazz lyric, You’ve got the right key but the wrong keyhole), and the lover hiding in the chest, in turn sexually suggestive of the woman’s sexual organ. Nevertheless, here it is really the lengthy dénoument with the husband’s sadistic verbal and physical revenge that is most important. He offers her three choices of death: to cut off her head off, to sweep the house with her hair, or to let her sit up till morning bearing candles to entertain seven guests: Három halál közül melyiket választod: Vaj föbe löjjelek, vaj fejedet veghem, Vaj hét asztal vendégnek vigon gyertyát tartasz” ‘Do you hear, wife, do you hear, wife, do you hear? Which one will you choose of the three deaths” ‘Do you hear, wife, do you hear, wife, do you hear? Which one will you choose of the three deaths” Have your choice now: shall I take your head? Or shall I sweep the house with your silk hair? Have your choice now: shall I take your head? Or shall I sweep the house with your silk hair? Or would you prefer to sit up till morn, And bear candle to the entrtainment of seven boards of guests’ The wife naturally falls into the trap of choosing the third option, which is in actuality the most sadistic one. The husband then orders the servants to roll her up head to toe in oilcloth covered in pitch and the batiste that was “given free” by his father-in-law (an illogical detail, since having turned back halfway, he would not have brought home the cloth), in effect making a candle out of her. In another version he has this done not by the servant but twelve young men, who are then to feast all night, with gypsy music playing, as she burns to death. Abstract Whereas Vargyas conjectures, probably without adequate justification, that the Hungarian version was a late addition to the tradition, based on a German ballad with circulated in the eighteenth century, Armistead (“Ballad” & “Hungary”: 70-71) has cogently argued for the importance in the Hungarian repertoire of the region’s interaction with neighboring traditions. The Hungarian ballad called “Bárcsai” by the lover’s name, is known only in Transylvania, in five versions and one fragment (English versions in Leader 232-39, Vargyas Hungarian Ballads 11-14, Balassa & Ortutáy 531-32). The fifty line Barcsai begins in direct dialogue, with the deceitful wife urging the husband to go to Kolozsvár to bring her back some batiste from her father’s house, when their son interrupts to warm the father not to leave because the mother loves Bárcsai: ne menj apám, ne menj, aj ne menj házrol ki: Anyámasszony bizony Bárcsait szereti ‘Do not go, father, do not go, aye, do not leave the house, / for mother is in love with Barcsai’. This beginning is unusual in that the wife does not merely use the opportunity of her husband being away but actively plots his departure, and even more unusual is active voice of the son because normally the mention of children in adultery stories is avoided, as their presence would be too suggestive that that child may not be the father’s either. Heeding his son’s words, the husband decides to turn back halfway. On his return he asks his wife to open the door, and there ensues a ritual series of requests and delaying tactics where she asks him, in turn, to wait until she puts on her skirt, her apron, her newly-soled boots, and her scarf. Here the ritual questions are not a testing dialogue about the signs of the other man, as in most of the variants, but rather a catalogue of the (presumably undressed) wife’s reverse striptease. Vargyas claims that there are no known analogues to this ballad, but this scene actually illustrates the clear interaction with other traditions, as, for example a Catalan version, where another adulterous wife who hears a knock says she is ready to open for her lover but would take time to put on her clothes and shoes if it is her husband (Vasvari Heterotextual 77, n. 4). Abstract As in the English predecessor, the husband sees, as he goes deeper and deeper into his house, from barn, to kitchen, to hall, to living room to room, to bedroom: three horses, which wife calls milk cows her mother sent three boots which she calls beer tankard three swords which she Although F. W. Meyer did translate “The Tenant Farmer’s Return” from English in l790 , which he called a Bänkersängerlied ‘street ballad’, the Hungarian version has enough difference to show that it has clearly entered oral tradition and been influenced by other variants. For example, in the German version the boots are beer tankards, the swords are spits to roast larks, and the jackets are tablecloths. As in the English predecessor, the husband sees, as he goes deeper and deeper into his house, from barn, to kitchen, to hall, to living room to room, to bedroom: three horses, which wife calls milk cows her mother sent, three boots, which she calls beer tankard, three swords which she calls spits for larks (Lerchenspitze), three jackets which she calls tablecloths, until he gets to the bedroom and in bed he sees three knights, whom she calls milkmaids; then comes the punchline: I’ve never seen milkmaids with moustaches (Zwickelbarte). In the Hungarian version the testing dialogue proceeds in five parts. The husband asks, in turn, whose are the boots, the swords (in the plural), the pair of soldiers’ hats, the coats, and he gets the replies that they are milk jugs, swords, kitchen knives, milk pots, and the servant girl’s mildewed dress. The wife’s replies are not merely lies but transparently ludicrous reinterpretations, of language, attempting to deceive the husband both verbally and visually to convince him, literally, not to believe his eyes. Finally, he asks, who are the pair of soldiers on his bed and she replies it is her grandmother’s maid sweeping the room, to which he snaps back what should be the punch line: szolgálonak pörge bajszá ‘ Aj, ki látott már (‘who ever saw maids with red moustaches’). Abstract As in the English predecessor, the husband sees, as he goes deeper and deeper into his house, from barn, to kitchen, to hall, to living room to room, to bedroom: three horses, which wife calls milk cows her mother sent, three boots, which she calls beer tankard, three swords which she calls spits for larks (Lerchenspitze), three jackets which she calls tablecloths, until he gets to the bedroom and in bed he sees three knights, whom she calls milkmaids; then comes the punchline: I’ve never seen milkmaids with moustaches (Zwickelbarte). In the Hungarian version the testing dialogue proceeds in five parts. The husband asks, in turn, whose are the boots, the swords (in the plural), the pair of soldiers’ hats, the coats, and he gets the replies that they are milk jugs, swords, kitchen knives, milk pots, and the servant girl’s mildewed dress. The wife’s replies are not merely lies but transparently ludicrous reinterpretations, of language, attempting to deceive the husband both verbally and visually to convince him, literally, not to believe his eyes. Finally, he asks, who are the pair of soldiers on his bed and she replies it is her grandmother’s maid sweeping the room, to which he snaps back what should be the punch line: szolgálonak pörge bajszá ‘ Aj, ki látott már (‘who ever saw maids with red moustaches’). p y Although F. W. Meyer did translate “The Tenant Farmer’s Return” from English in l790 , which he called a Bänkersängerlied ‘street ballad’, the Hungarian version has enough difference to show that it has clearly entered oral tradition and been influenced by other variants. For example, in the German version the boots are beer tankards, the swords are spits to roast larks, and the jackets are tablecloths. As in the English d h h b d h d d d i hi h f b Although F. W. Meyer did translate “The Tenant Farmer’s Return” from English in l790 , which he called a Bänkersängerlied ‘street ballad’, the Hungarian version has enough difference to show that it has clearly entered oral tradition and been influenced by other variants. For example, in the German version the boots are beer tankards, the swords are spits to roast larks, and the jackets are tablecloths. Abstract This very sadistic version shows that the husband’s performance of heterosexual masculinity – here the salvaging of his honor -- has to be acted out in a disciplinary program, whereby males need to show that they can master their wives in front of other men in a homosocial bonding situation, in this case specifically with young unmarried men, who are to learn from it how to deal with insubordinate women (for a similar case see my analysis of Dec 9.9, in “Buon cavallo”). Alongside this sadistic Bárcsai there exist comic adultery ballads in Hungarian, where it is less the craftiness of the woman that is foregrounded than the stupidity of the henpecked and cuckolded husband. In a one-hundred verse ballad collected by Béla Bartok from a twenty-six year old woman in 1918, A megcsalt férj ‘the deceived husband’ (Vargyas, A Magyar népballada, 936-38) the husband is so aware of his plight that that as he goes deeper and deeper into the house, from the barn, to the kitchen, hall, the main room, and the bedroom, he keeps on repeating: Ehagyatott, megcsalhatott / férj vagyok mindég (‘Am I not a silly, deceived husband I have ever been?’). The couple 4 4 exchange incongruously endearing vocatives during their interchange, he beginning each questions with the ironic [kérdem] edes kedves felesegem ‘[I ask you] my dear sweet wife’ and she, in turn, replying mi baj van, angyalom ‘what’s wrong my angel’, a detail probably translated from a German version, which has herzliches Weibchen and mein Schätzchen, respectively. z , p y Although F. W. Meyer did translate “The Tenant Farmer’s Return” from English in l790 , which he called a Bänkersängerlied ‘street ballad’, the Hungarian version has enough difference to show that it has clearly entered oral tradition and been influenced by other variants. For example, in the German version the boots are beer tankards, the swords are spits to roast larks, and the jackets are tablecloths. Az Ablaknál ki járt éppen? Abstract // No, it certainly wasn’t the cat but it was your lover himself. Sári lányom, ki járt itt, ki járt itt,/ kinek a nagy pipaszára maradt itt? Gépész, uram, jára itt, jára itt. / Annak a nagy pipaszára maradt itt. Hát az ágyat mi lölte, mi lölte,/ Mért van ugy összegyürve, de gyürve? Cica fogott egeret, egeret, / Jatszott vele eleget, eleget. Hát a hasad mi lölte, mi lölte, / Miert van igy megpüffedve, püffedve? Túrot ettem fikhagymát, fikhagymát, / Attol püffedt ekkorát, ekkorát. Sári lányom, ki járt itt, ki járt itt,/ kinek a nagy pipaszára maradt itt? Gépész, uram, jára itt, jára itt. / Annak a nagy pipaszára maradt itt. Hát az ágyat mi lölte, mi lölte,/ Mért van ugy összegyürve, de gyürve? Cica fogott egeret, egeret, / Jatszott vele eleget, eleget. Hát a hasad mi lölte, mi lölte, / Miert van igy megpüffedve, püffedve? Túrot ettem fikhagymát, fikhagymát, / Attol püffedt ekkorát, ekkorát. (‘ My dear Sarah, who was here, who was here, / whose big pipe stem got left behind? / A mechanic was here, my husband, / it was his big pipe stem that got left behind. // And the bed, who poked at it, who poked at it? / Why is it wrinkled, all wrinkled?/ The kitten caught a mouse, a mouse / and played with it a lot, a lot./ And what poked at your stomach, what poked it? / Why is it so puffed up, puffed up? / I ate farmer cheese with garlic, / and that’s why it puffed up so much, so much.) In the nineteenth century there were also a number of songs collected in French and Italian dialects that had comic dénouments. However, by far the most widespread comic versions are in Anglophone tradition, in two main variants, Our Goodman and the Merry Cuckold, , where the song is a folk classic, perhaps the most popular ballad in oral tradition. Over four hundred versions have been collected in British and American tradition, including many from hillbilly recordings, and as far away as New Zealand and even India. Because of its erotic nature Francis James Child only exceptionally allowed it into his magnum collection, according it “brusque disrespect” (Child V. 274). Abstract At this point, the comical version should end, or at best continue with an appropriate revenge, as in the German version, where he beats his wife, claiming they were caresses sent by her mother, which Vargyas deemed to be a very ”felicitous ending,” but which I have called elsewhere “bawdy battering,” that is, where a wife’s beating is considered the height of humor because it is embedded in a comic text (Vasvari “Buon cavallo”). However, here the ending suddenly turns brutal, with the husband declaring that he will hold a ball and behind the door there awaits a knotted rope and a big cudgel, an ending that is clearly a contamination from Bárcsai. The knotted rope also recalls the Hungarian proverb: a kötél ásztatva, az asszony verve jó (‘ a rope is good when wet and a woman when beaten’). Nor can this sadistic version simply be a confused version recounted by one person because it appears in another version as well. This is a clear indication that even if this comic version is a translation from the German, the deep structure adultery plot is so primary that it is possible to fuse such disparate treatments. li i i h i d di i h b f In living twentieth-century Hungarian and Roma tradition there are a number of comic songs about adulterous wives reduced only to the bare-bones testing dialogue, which I offer here in translation. The first is translated from Roma (Csikó, Csenki) and the second was collected by Bártok and Kodály, A magyar népzene tára, no. 454). We should not be surprised by the image of Puss in Boots in the first verse, if we consider its potential erotic suggestiveness: Az Ablaknál ki járt éppen? 5 5 Mondd meg nekem, feleségem! Ablakom alatt ki járt ma? Csak a szomszédok macskaja, Szomszédunké ottan hátra, Szomszédunké ottan hátra. De macskának nincs kalapja, A macskának nincs kalapja, Se a lábán nincsen csizma, Se a lábán nincsen csizma. Ej, dehogy a macska volt az, A szeretöd maga volt az. ‘Who just walked by under the window /Tell me, my wife! / /Who walked under the window today? / Just the neighbor’s cat, / our neighbor’s back there, / our neighbor’s back there. // But cats don’t wear a hat, / cats don’t wear a hat, / and they don’t wear boots either,’ and don’t wear boots either. Abstract Our Goodman is better classified as a joke ballad, a narration of a humorous event working towards the surprise punch line, so that if the punch line is omitted or ruined the story is ruined, which explains why some Spanish and Hungarian versions with the violent endings grafted on cannot work except for those who find the wife battering hilarious. In a broader sense, Our Goodman belongs to jokelore, a liminal popular oral discourse, the vast majority of whose manifestations are male-to-male, commenting on gender relations 6 and aimed at relieving unfulfilled or failed sexual desire, with blame always placed on woman as, alternately, promiscuous or frigid (Dreitser). In Our Goodman the husband comes home drunk every night, a detail omitted in the The Merry Cuckold, where, on the other hand, the extra ludicrousness is provided by the husband coming home and finding not one but three men in his bed. Both proceed with versions of the expected testing dialogue, where the wife gives absurd replies, claiming that the horse is a pig, or that the three horses are milking cows, the sword[s] are a porridge-spurtle or roasting spits, the wig is a hen, or, alternately, that the three cloaks are bedspreads, the boots pudding bags, the three pairs of breeches, petticoats, the three hats, skimming dishes, and the head of the lover in bed, a mush melon, or the three lovers in bed, milk-maids. Punch lines include: Godzounds! Milking maids with beards on! The like was never known! or, in another version, But whiskers on a baby’s face / I never saw before, or the somewhat more snappy I’ve traveled this wide world over, a hundred miles or more, / but whiskers on a mush melon I never did see before (with the Yiddish variant, But whiskers on a cabbage head I never saw before). In fact, in many cases informants were handicapped by modesty and would not reveal much more obscene versions they knew, and in other cases collectors would not write down such versions (on the bowdlerization and expurgation of sexual folklore, see Goldstein: 374). Abstract Fortunately we have a few of these collected in Eugene Vance’s collection of “unprintable folk songs,” which give a flavor of some of those bowdlerized punch lines, as in (53-57; see also Goldstein, 376): But a pecker on a hired girl/ I never seen before; But ballyx on a rolling pin/ I never seen before, or to the question: Whose prick is in the cunt where mine had ought to be? the following answer: You old fool, you blind fool, an’ can’t you plainly see It’s nothin’ but a parsnip my granny sent to me?— You old fool, you blind fool, an’ can’t you plainly see It’s nothin’ but a parsnip my granny sent to me?— It’s nothin’ but a parsnip my granny sent to me?— Well, its’ miles I have traveled, this wide world all o’er, Well, its’ miles I have traveled, this wide world all o’er, But ballyx on a parsnip I never saw before. But ballyx on a parsnip I never saw before. while in the tamer versions the lover is metonymically represented by his beard of whiskers, which is, along with face, forehead, eyes, and mouth, the public part of his face which concentrates social identity, in the obscene versions he is metonymically reduced to his private parts, which normally are concealed. Worse, these sex organs are described as parsnips, or in another version, pickled beets, what I have elsewhere dubbed “vegetal- genital onomastics.” As I was preparing this study I happened to receive an e-mail version of jokelore that in its deep structure clearly belonged to this same tradition, but with a difference, for here for the first time we get a husband who gives more than ample cause for his wife’s 7 adultery, and it is she who gets the appropriate punch line, as well. Could this finally be a subversive version in oral jokelore of our tired retold tale? I offer here a translation of the Hungarian version I heard, but I assume that English versions circulate, as well: A wife says to her husband in a dissatisfied voice: -- Géza! The light isn’t working. -- What am I, an electrician? The next day she says again. -- Géza, the faucet is leaking. -- What am I, a plumber? The third day. The third day. -- Géza! The shelf fell off. -- What am I, a carpenter? Abstract The next day the husband steps into the house, and the lights are lit, the faucet doesn’t drip, even the shelf is in its place. -- Who fixed these? -- The neighbor came over and he said he’d fix everything, if I bake him a cake or I go to bed with him. -- And what did you do? -- What am I, a baker? In a continuation of the spirit of the medieval discourse on women, where the earliest versions of these tales originated, some scholars today are still capable of misreading misogynist texts as if they were guidebooks for morality. Wolfgang Spiewok (xii) raises the question why might it be that so many German novelle ridicule cuckolds, and why in a society where a husband would have had the legal right to kill both the wife and lover, they escape without punishment. He concludes that these stories were a warning to married men, and that, after all, these “cheerful and amusing” stories can be useful to the modern Leser ‘[male] reader’ who, if he finds himself in the role of the husband, will be able to learn some lessons, which today are still full of life and worthy of being read. In contrast to such masculinist misreading, in a perceptive study Rüdiger Schnell (776-82) discusses how the most important boundary in discourses on the sexes is not between positive and negative representations of women, as both are the result of the same andocentric perspective aimed at the domestication and repression of women. What is more important is the distinction between the discourse on woman and the discourse on marriage, where in the former images of women are constructed without any critical reflection about men, as in all but the very last jokelore example of my study. In contrast, the discourse on marriage discussed the difficulties of married life and presents a more differentiated view of women. 8 8 BIBLIOGRAPHY Armistead, S. G. “Ballad” & “Hungary”. In C. Lindahl et al eds. Medieval Folklore. An Encyclopedia of Myths, Legends, Tales, Beliefs and Customs. Santa Barbara, CA: Lindow Editors: 61-2, 71-2, 2000. Balassa, Iván & Ortutáy, Zoltán. Hungarian Ethnography & Folklore. Budapest: Korvina Kiadó., 1984. Bartók, Béla. l979 [1931 A magyar népdal. ]. Hungarian Folk Music. trans. M. D. Calvocorressi. Westport, Conn: Hyperion P., 1979 [1931]. Bartók, Béla & Zoltan Kodály. eds. A magyar népzene tára. I.1. Budapest: Akadémiai Kiadó, 1973. Child, Frances James, ed. 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Roll Me in Your Arm: Unprintable Ozark Folksongs & Folklore.I. Fayeteville: U of Arkansas P., 1992. Vargyas, Lajos, ed. A magyar népballáda és Europa II. Budapest: Zenekiadó, 1976.. Vargyas, Lajos, ed. A magyar népballáda és Europa II. Budapest: Zenekiadó, 1976.. ____________. ed. Balládas könyv. Budapest: Zenemükiadó, 1979.. ____________. ed. Hungarian Ballads and the European Ballad Tradition. 2 vols. Budapest: Akadémiai Kiadó, 1983. Vasvári, Louise O. The Heterotextual Body of the Mora Morilla. London: Queen Mary and Westfield College, l999. ______________. “Buon cavallo e mal cavallo vuole sprone, e buona femina e mala femina vuol bastone’: Medieval Cultural Fictions of Wife Battering.” In Albrecht Classen, ed. Love, Marriage, and Transgression in Medieval and Early Modern Literature. Tempe, AR: Arizona Center for Medieval and Renaissance Studies, 2004: 313-336. ______________. “Cunningly Lingual Wives in European Ballad Tradition.” In Adrienne Martin & Cristina Martinez Carazo, eds. Spain’s Multicultural Legacies, Juan de la Cuesta, 2008 [in press]. 10
https://openalex.org/W2412510786	https://scindeks-clanci.ceon.rs/data/pdf/0354-8422/2014/0354-84221470008S.pdf	Croatian	null	Negativne pojave u kolektivu i preventivne mere (alkoholizam, narkomanija, sekte i suicid)	Sestrinska reč/Sestrinska reč	2,014	cc-by	2,432	Cilj nege j g - Sluzokoža usne duplje neće biti izmenjena do kraja hospitalizacije. j g - Sluzokoža usne duplje neće biti izmenjena do kraja hospitalizacije. Obezbediti propagadni materijal u vidu brošura, da paci jentima i porodici budu dostupne, da samim tim mogu videti šta im nije jasno, i tako tačno znati šta treba da pitaju. - Crvenilo kože će se povući do kraja hospitalizacije. - Neće doći do nastanka infekcije IVK do kraja hospitalizacije. - Pacijent će biti obučen za samostalno hranjenje preko gastro stome do polaska kući. Obezbediti edukativne programe u vidu demostracije za sestre koje ne rade na odeljenju hirurgije, a zbog potrebe posla treba da znaju i rade. - Pacijent će dobiti sve informacije u vezi sa gastrostomom do kraja hospitalizacije. STRU^NA PRAKSA STRU^NA PRAKSA ZAKLjUČAK - Pacijent će imati bolji i kvalitetniji san do polaska kući. Sestra je podrška i oslonac prvenstveno bolesniku, a potom i porodici. Ona je tu da pomogne savetom, razgovorom, toplom ljudskom reči i naravno demostracijom. Sestra mora prepoznati nivo i mogućnost usvajanja znanja, tako da informacije što ad ekvatnije približi pacijentovom obrazovnom nivou. Cilj je da informacije budu razumljive bolesniku. - Porodica pacijenta će biti obučena i dobiće sve potrebne infor macije u vezi gastrostome. Literatura 1. Dragović M., Gerzić Z.; Osnovi hirurgije 2. Terzić N.: Zdrastvena nega u hirurgiji, Beograd, 2006. 3. Đurica S. Interna medicina, Beograd, 2000. 4. Petrović,Z.: Bolesti jednjaka,zeluca i duodenuma; 5. Pesko P,, Ranđelovic T,, Simic A.,Bjelovic, M., Stojkov D. Univerziteta u Beogradu, Beograd, 2008. 1. Dragović M., Gerzić Z.; Osnovi hirurgije 2. Terzić N.: Zdrastvena nega u hirurgiji, Beograd, 2006. mic A.,Bjelovic, M., Stojkov D., Sabljak P.:Hirurgija ezofagogastroduodenuma.Hirurgija za student medicine.Medicinski fakultet Stru~ni rad Evaluacija - Sluzokoža usne duplje je normalnog izgleda i vlažnosti. - Crvenilo kože se povuklo do planiranog dana otpust Aktivno učenje je metod u kome učesnici aktivno učestvuju u čitavom procesu učenja, donošenjem odluka i praktičnom osposobljavanju, da naučeno primene kroz ponašanje u sva kodnevnom zivotu. Aktivno učešće je snažna motivacija za njegovu praktičnu primenu. - Nije došlo do infekcije IVK do kraja hospitalizacije. - Pacijent je usvojio i savladao veštinu hranjenja, i - Porodica je efikasno prilagođena zdravstvenom stanju svog člana porodice. ALKOHOL Od somatskih najčešće su masna jetra, ciroza, ca jetre, oboljenja or gana za varenje, oštecenje perifernih nerava, poremecaj hormonskog statusa, oboljenje KVS. Alkohol remeti sve psihičke funkcije, pažnju, volju, upamćivanje, koncen traciju, dovodi do depresije, anksioznosti, poremećaja ličnosti (asocijalnog, emocionalno nestabilnog). Nakon višegodisnjeg konzumiranja alkohola nastaju delirijum tremens, korsakovljeva psihoza, alkoholna epi i patološka ljubomora. somatske (telesne ) i psihicke. NARKOMANIJA Narkomanija je bolest savremenog čoveka, označava stanje potrebe za uživanjem u psihoaktivnim supstancama tj pojedi nac je zavistan od droge. Narkomani su osobe koje uživaju drogu tj osobe kod kojih je zbog kontinuirane upotrebe droge poremećeno funkcioni sanje u svim sferama zivota, ličnog, profesionalnog, porodičnog i socijalnog. Narkomanija najšesće počinje u adolescentnom periodu, javlja se u disfunkcionalnim porodicama gde je već prisutan problem zavisnosti. Droga remeti normalno bio-psiho- socijalno sazrevanje mlade ličnosti. Dovodi do trajnog oštećenja CNS, javljaju se zatim duševni poremećaji (depresije, psihoze, anksioznosti), formira teže poremećaje licnosti (asocijalne, emocionalno nestabilne). Droga omogućuje pridruživanje teških telesnih bolesti kao sto su HIV, Hepatitis B, C i endokarditis. Narkomani napuštaju školovanje, nezaposleni su, čine razna krivična dela: kradu, prostituišu se, preprodaju drogu, otimaju, pljačkaju, ubijaju se ili ubi jaju druge. Alkoholizam je bolest koja nastaje zbog nekontrolisane i redovne upotrebe alkoholnih pića. VMC Karaburma Beograd Sažetak: Kolektiv predstavlja grupu ljudi udruženu radom radi zajedničkih ciljeva. Negativne pojave u kolektivu i direktno, oštecuju somatsko i psihičko zdravlje, i indirektno, stvarajuci konflikte, utiču na pojedinca i onemogućuju njegovo funkcionisanje u svim sfera ma zivota. Konflikti su praćeni psihičkom napetošću, uznemirenošću, izazivaju stres i depresiju sto dovodi do pojave psihosomatskih poremećaja. Posledice su velike: brojna bolovanja, odsustvovanja sa posla, gubitak radne sposobnosti, beg iz kolektiva, suicid. Sve ovo neminovno dovodi do dezorganizacije rada u kolektivu. Zbog toga se sprovode preventivne mere. U radu su izneta naša iskustva u ranom otkrivanju, kontroli i rešavanju eventualnih negativnih pojava u vojnoj U radu su izneta naša iskustva u ranom otkrivanju, kontroli i rešavanju eventualnih negativnih pojava u vojnoj sredini. Cilj rada je da se prikaže prevencija nastanka negativnih pojava i značaj edukacije zaposlenih. Ključne reči: alkoholizam,narkomanija, sekte, suicid, prevencija Abstract: Collective is a group of people who work together to achive a common objective. Negative phenomena in the collective direc thy damage somatic and psychological health and indirecthy the occurance of conflicts afffect the individual discrupting functioning in all spheres of life. Conflicts caused by stress and depression are accompanied bypsychological sunspence and anxiety which head to the occurance of psychosomatic disorders. The consequences are severe: many sick days,absence from work, loss of working capacity, escape from the collective, suicide. ll this inevitably leads to desorganization of work in a group. This paper present our experiencies in the early detection, control and solving negative effects in the military service. The aim of this work is to present preventive measures, actually the prevention of negative efects and the importance of education of the This paper present our experiencies in the early detection, control and solving negative effects in the military service. The aim of this work is to present preventive measures, actually the prevention of negative efects and the importance of education of the employees. per present our experiencies in the early detection, control and solving negative effects in the military service. h p p p p y g g ff y The aim of this work is to present preventive measures, actually the prevention of negative efects and the importance of education of the employees. Keywords: alcoholism, drog addiction, suicid, sect, prevention 9 STRU^NA PRAKSA UVOD reći da alkohol šteti radnom uspehu, deluje štetno na moral i polet drugih u radnoj sredini. U manjoj količini deluje na kon trolu i norme ponašanja, u većoj usporava reflekse, menja nor malno misljenje i ošamucuje svest. U znatno većoj količini dovodi do nesvesnog stanja, ponekad i do smrti. N N ajčešće negativne pojave u kolektivu su alkoholizam, nar komanija, sekte i suicid. Sve ove pojave dovode do oštećenja psihičkog i somatskog zdravlja, do problema u porodici i na radnom mestu. U svakom slučaju alkoholizam, narkomanija, sek te i vojna sredina su nekomplementarni, što se reguliše zakonskim merama i merama koje su propisane u pravilu službe. Iako se iste sprovode u vojnom kolektivu, ipak se jave negativne pojave i tada se preduzimaju adekvatne mere (primarna, sekundarna i tercijalna prevencija) da ne bi došlo do oštećenja radne sposobnosti i do smrti zaposlenih. Da bi prevencija bila uspešna potrebno je naučiti što više o negativnim pojavama . ALKOHOLIZAM Alkoholizam je bolest koja nastaje zbog nekontrolisane i redovne upotrebe alkoholnih pića. Alkoholičar je osoba koja nije u stanju da kontrolise pijenje alkoholnih pica. Takva osoba ima probleme u porodici, na rad nom mestu i sa samim sobom. Alkohol uništava osobu koja pije, remeti joj život, uništava samopoštovanje i poštovanje od strane drugih ljudi, oštecuje joj zdravlje i skraćuje život. S druge strane alkohol uništava porodicu, dovodi do siromaštva, razvoda, kriminala, samoubistva ili ubistva, stvara konflikte, a samim tim onemogućuje normalan razvoj dece. Alkoholičari nedovoljno vode brigu o deci i ne razmišljaju o njihovoj buducnosti. Alko hol na radnom mestu smanjuje efikasnost na poslu i dovodi do lažnog osećanja boljeg rada, povećava broj nesreća na poslu, a alkoholičar dovodi u opasnost ostale iz svoje okoline. Može se POSLEDICE ALKOHOL PSIHOTIČNI POREMEĆAJI OSLABLJEN VID OŠTEĆENJA ZUBA KATAR JEDNJAKA PRESTANAK DISANJA GASTRITIS ČIR NA ŽELUDCU SRČANA OBOLJENJA OBOLJENJA JETRE OŠTEĆENJA KOŽE IMPOTENCIJA NEPLODNOST PANKREATITIS OSTEOPOROZA KANCER POSLEDICE ALKOHOL PSIHOTIČNI POREMEĆAJI OSLABLJEN VID OŠTEĆENJA ZUBA KATAR JEDNJAKA PRESTANAK DISANJA GASTRITIS ČIR NA ŽELUDCU SRČANA OBOLJENJA OBOLJENJA JETRE OŠTEĆENJA KOŽE IMPOTENCIJA NEPLODNOST PANKREATITIS OSTEOPOROZA KANCER Alk h li j b l k j j b k li i POSLEDICE ALKOHOL PSIHOTIČNI POREMEĆAJI OSLABLJEN VID OŠTEĆENJA ZUBA KATAR JEDNJAKA PRESTANAK DISANJA GASTRITIS ČIR NA ŽELUDCU SRČANA OBOLJENJA OBOLJENJA JETRE OŠTEĆENJA KOŽE IMPOTENCIJA NEPLODNOST PANKREATITIS OSTEOPOROZA KANCER SUICID Suicid je namerno i svesno, aktivno ili pasivno oduzimanje vlastitog života. Tu se ubrajaju i pokušaji oduzimanja sopst venog zivota i sve vrste samopovređivanja. Faktori rizika za suicid su: psihijatrijski poremećaj, soci jalni problemi, porodični, biološki faktori, somatska oboljenja. Od psihijatrijkih poremećaja najčešći su bipolarni afektivni poremećaj i to depresivna faza, zatim Shizophrenia, emociona lno nestabilne ličnosti, poremećaj ličnosti-asocijalni i granični tip, adolescentna kriza, panični poremećaji i PTSP. Veliki broj sa moubistva desava se kod PTSP, tj kod stresnih životnih događaja, npr kod gubitka voljene osobe, velikog materijalnog i emotivnog gubitka. Zatim kod žrtve zlostavljanja (fizičkog, psihičkog ili seksualnog), žrtve mobinga i u ratu. Od socijalnih faktora na jzastupljeniji su siromaštvo, nezaposlenost i migracija. Od bioloških napomenućemo smanjenu koncentraciju serotonina u likvoru. Kada govorimo o porodičnim faktorima spomenuc emo porodičnu predispoziciju i probleme u porodici. Somatska oboljenja kada su udružena sa nepodnošljivim bolovima pred stavljaju rizik za samoubistvo. Tu spadaju karcinomi, oboljenja CNS, EPI , MS, demencija, sida, ciroza jetre, porfirija. NPS U VMC KARABURME - Selekcija kandidata za srednje vojne škole , više škole i akademiju; - Selekcija kandidata za profesionalnu vojnu službu; - Selekcija kandidata za školovanje u inostranstvu; - Selekcija kandidata za mirovne misije; - Selekcija kandidata za mirovne misije; - Selekcija kandidata za stipendiranje učenika i studenata; - Selekcija kandidata za Generalštabne i Komandnoštabne skole; - Praćenje mentalnog zdravlja pripadnika VS i članova njihovih porodica; - Pružanje savetodavne pomoći starešinama iz vojnomedicinske higijene; - Timsko angažovanje na poslovima rane dijagnostike, terapije i ocene sposobnosti pripadnika VS. Prvi znaci upozorenja su kada suicidalna osoba počinje da se usamljuje, udaljava od porodice i prijatelja i krene da govori o samoubistvu. Ove osobe imaju poremećaj spavanja, uznemi rene su, imaju naglu promenu raspoloženja, izliva besa, pokušaj suicida, poneki upotrebljavaju alkohol, lekove ili droge. Osobine koje su zajednicke svim samoubicama su impulsivnost, ambiva lencija i rigidnost. Najveci broj suicidalnih ne želi da umre tako da pokušaj suicida treba shvatiti kao apel za pomoć, znak da nešto treba menjati u životu tih ljudi. SEKTE Sekte su organizovane religijske grupe predanih vernika zarad zajedničkih interesa. Sve sekte se bave destruktivnom manipulaci jom ljudi da bi ih iskorištavale. U sektu se ulazi dobrovoljno, ali teško izlazi. Protokoli lečenja su kao kod lečenja bolesti zavisnosti. Alkoholičar je osoba koja nije u stanju da kontrolise pijenje alkoholnih pica. Takva osoba ima probleme u porodici, na rad nom mestu i sa samim sobom. Alkohol uništava osobu koja pije, remeti joj život, uništava samopoštovanje i poštovanje od strane drugih ljudi, oštecuje joj zdravlje i skraćuje život. S druge strane alkohol uništava porodicu, dovodi do siromaštva, razvoda, kriminala, samoubistva ili ubistva, stvara konflikte, a samim tim onemogućuje normalan razvoj dece. Alkoholičari nedovoljno vode brigu o deci i ne razmišljaju o njihovoj buducnosti. Alko hol na radnom mestu smanjuje efikasnost na poslu i dovodi do lažnog osećanja boljeg rada, povećava broj nesreća na poslu, a alkoholičar dovodi u opasnost ostale iz svoje okoline. Može se U celom svetu ih ima preko 2000. Vođe verskih sekti teze jed nom cilju, a to je materijalno bogatstvo. Promovišu skromnost, a žive u izobilju, ne biraju sredstva da dođu do cilja. Verske sekte utiču na psihičko zdravlje svojih sledbenika. Dovode do destrukcije, asocijal nog ponašanja, autoagresivnog samopovređivanja ili samoubistva, seksualne izopačenosti, silovanja, otmice, ubistva. Verske sekte su ko mercijalno-profitabilne organizacije sa političkom, pseudonaucnom, kriminalnom, psihoterapeutskom i vojnom pretenzijom. Sekte raz 10 STRU^NA PRAKSA SESTRINSKA PRAKSA araju telesno i duhovno zdravlje pojedinca, dezorganizuju pojedinca ,porodicu, kolektiv, društvo i državu. TERCIJALNA PREVENCIJA ima za cilj da spreči radnu nespo sobnost i invaliditet i pristupa lecenju, rehabilitaciji i socijalnoj adaptaciji. TERCIJALNA PREVENCIJA ima za cilj da spreči radnu nespo j p p sobnost i invaliditet i pristupa lecenju, rehabilitaciji i socijalnoj adaptaciji. sobnost i invaliditet i pristupa lecenju, rehabilitaciji i socijalnoj adaptaciji. Potrebno je edukovati zaposlene kada da se obrate psiho logu i psihijatru. Ukoliko se pojedinac nalazi u kriznoj situaciji a nije u stanju da reši i prevaziđe samostalno. Ukoliko je prob lem u adaptaciji, ako ima porodične i bračne probleme, adoles centne probleme, probleme u komunikaciji neophodno je da se javi na pregled psihijatru. Prevencija negativnih pojava Preventivni rad se ogleda u vidu savetovanja, držanja tematskih predavanja i edukacije. Rade se sistematski pregledi redovni i vanredni (ukoliko je starešina, trupni lekar ili psiholog primetio promenu u ponašanju). Sve ove mere na vreme otkrivaju mentalne poremećaje i na taj način sprečavaju vanredne dogadjaje. U Srbiji ima 300 000 alkoholicara, 150 000 narkomana, 1500 suicida godišnje, a u Vojsci se godišnje u proseku ubi je 4 pripadnika (sto je u poređenju sa 1500 mali procenat). Zanemarljiv je i broj lečenih narkomana (oko 30 u VS sto je zanemarljivo u odnosu na 150 000). Nešto je veći broj lečenih alkoholičara ali u poređenju sa brojem alkoholicara u Srbiji opet je zanemarljiv. Kapital jednog kolektiva je zdravlje zaposlenih i zbog toga se sprovode preventivne mere da bi se sačuvalo i unapredilo zdravlje zaposlenih. PRIMARNA PREVENCIJA, pre stupanja na radno mesto neophodno je kontrolisati psihičko i somatsko zdravlje kandi data. Ali ukoliko primetimo da u kolektivu postoje ljudi koji se parniče, koji su hronični svadljivci, pripadnici sekti, suicid alni potrebno ih je poslati na vanredni sistematski pregled uz mišljenje starešine i nalaza psihologa iz jedinice. Neophodno je da na radnom mestu rešavamo konfliktei uspostavljamo kon struktivne interpersonalne odnose. Na taj način unapređujemo mentalno zdravlje. Jako je važno edukovati zaposlene i propa girati zdrave stilove života. Predrasude o neizlečivosti duševnih poremećaja i drugih zabluda u vezi duševnih poremećaja treba razbiti. Razlog ovako malog broja alkoholičara, narkomana i suicid alnih u vojnom kolektivu je u dobro organizovanoj prevenciji. SEKUNDARNA PREVENCIJA ima za cilj da spreči SEKUNDARNA PREVENCIJA ima za cilj da spreči hronicni razvitak bolesti i to je u domenu psihijatra. Lečenje je uspešnije ako se poremećaj prepozna u samom početku, di jagnostifikuje i što pre se krene sa lečenjem, da poremećaj ne dovede do invaliditeta ili smrti. 1. Stojiljković S. (1986) Psihijatrija sa medicinskom psihologijom, Medicinska knjiga Beograd-Zagreb 2. Kecmanović D.(1980) Psihijatrija, Medicinska knjiga Beograd-Zagreb 3. Kaličanin P. (2002) Psihijatrija (Specijalni deo II tom)Elit Medica, Beograd 4. Petrović Stevan (2003) Droga i ljudsko ponašanje, peto dopunjeno izdanje, Partenon Beograd 5. Medjunarodna klasifikacija bolesti i uzroka smrti, SZO ZAKLJUČAK U svakom kolektivu postoje negativne pojave. Pojave su prisutne i u vojnom kolektivu. Program sprovodjenja mera zaštite i unapređenja mentalnog zdravlja zasniva se na savre menim naučnim dostignućima iz oblasti socijalne psihijatrije, mentalne higijene i psihologije. Ova zaštita uskladjuje se sa građanskom zdravstvenom službom. U poređenju sa drugim kolektivima u vojnom je procenat negativnih pojava mnogo manji, a za to je zaslužna prevencija. Na kraju može se reci da nije svejedno sprečiti negativne pojave ili lečiti njihove kom plikacije. Literatura 11 11
https://openalex.org/W4386832131	https://www.scielo.br/j/delta/a/DPbmRPcwdgqQ8X97VGjLkCR/?lang=pt&format=pdf	Portuguese	null	A sessão de psicanálise como gênero do discurso: enquadre, terapia e encontro de vozes em visada bakhtiniana	DELTA. Documentação de estudos em lingüística teórica e aplicada/DELTA. Documentação de estudos em linguística teórica e aplicada	2,023	cc-by	10,074	This content is licensed under a Creative Commons Attribution License, which permits unrestricted use and distribution, provided the original author and source are credited. Artigos A sessão de psicanálise como gênero do discurso: enquadre, terapia e encontro de vozes em visada bakhtiniana Psychoanalysis session as a speech genre: setting, therapy and social voice tensioning through a Bakhtinian perspective A sessão de psicanálise como gênero do discurso: enquadre, terapia e encontro de vozes em visada bakhtiniana Psychoanalysis session as a speech genre: setting, therapy and social voice tensioning through a Bakhtinian perspective Eduardo da Silva Moll1 Maria da Glória Corrêa di Fanti2 Kelli Machado da Rosa3 https://dx.doi.org/10.1590/1678-460X202339354502 https://dx.doi.org/10.1590/1678-460X202339354502 1. Pontifícia Universidade Católica do Rio Grande do Sul (PPGL/PUCRS). Rio Grande do Sul – Brasil. https://orcid.org/0000-0002-0635-9845. E-mail: eduardosilva.moll@ gmail.com 2. Pontifícia Universidade Católica do Rio Grande do Sul (PPGL/PUCRS). Rio Grande do Sul – Brasil. https://orcid.org/ 0000-0002-5399-5377. E-mail: gloria.difanti@pucrs.br 3. Universidade Federal do Rio Grande (PPGL/FURG). Rio Grande do Sul – Brasil. https://orcid.org/0000-0002-6664-4912. E-mail: klro.rib@gmail.com RESUMO Embora pouco debatido na área dos estudos bakhtinianos, um marco da produção teórica do Círculo de Bakhtin foi a crítica de Volóchinov ao freudismo. Refletindo criticamente sobre o posicionamento do autor acerca da psicanálise, este artigo objetiva investigar o pequeno acontecimento 1. Pontifícia Universidade Católica do Rio Grande do Sul (PPGL/PUCRS). Rio Grande do Sul – Brasil. https://orcid.org/0000-0002-0635-9845. E-mail: eduardosilva.moll@ gmail.com 2. Pontifícia Universidade Católica do Rio Grande do Sul (PPGL/PUCRS). Rio Grande do Sul – Brasil. https://orcid.org/ 0000-0002-5399-5377. E-mail: gloria.difanti@pucrs.br 3. Universidade Federal do Rio Grande (PPGL/FURG). Rio Grande do Sul – Brasil. https://orcid.org/0000-0002-6664-4912. E-mail: klro.rib@gmail.com D.E.L.T.A., 39-3, 2023 (1-27): 202339354502 Eduardo da Silva Moll, Maria da Glória Corrêa di Fanti, Kelli Machado da Rosa social psicanalítico como um gênero do discurso em sua especificidade terapêutica, considerando o entrecruzamento de vozes constitutivo desse evento. Com uma pesquisa bibliográfica, recuperamos os pressupostos de Bakhtin e o Círculo que embasam o construto de gênero do discurso e investigamos o papel do enquadre na terapia psicanalítica freudiana. Conceitos como o de arquitetônica e de heterodiscurso para o Círculo, assim como o de resistência e de transferência para Freud ajudaram a explorar o lugar do setting na terapia. Defendemos que a relação discursiva psicanalítica especifica-se pela possibilidade de fazer da interação discursiva um espaço-tempo aberto ao diálogo inconclusível próprio do encontro terapêutico e da palavra em elaboração. Palavras-chave: interação discursiva; gêneros do discurso; heterodis- curso; sessão de psicanálise. 4. A expressão Círculo de Bakhtin foi criada pelos pesquisadores da área para designar o grupo de pensadores com formações e interesses distintos que se reuniram em Nével, Vítebsk e Leningrado, na Rússia, entre os anos de 1919 e 1929. Na área dos estudos do discurso, as contribuições de Bakhtin, Volóchinov e Medviédev orientam pesquisas em torno da relação entre sujeito, linguagem, arte e sociedade. Embora os escritos de Bakhtin a partir de 1930 desvinculem-se formalmente das reuniões do Círculo, nestes reconhecemos forte afinidade com as discussões do grupo, o que permite considerá-los também como do pensamento do Círculo (Barbosa & Di Fanti, 2020). 5. A dinâmica de trabalho de Volóchinov, que envolvia a publicação de um artigo e sua expansão em obra pouco tempo depois, atestada pela recuperação dos registros acadêmicos do autor no Instituto da História Comparada das Línguas e Literaturas do Ocidente e do Oriente - ILIAZV (Grillo, 2019), orienta-nos a creditar a autoria dos textos do freudismo a Volóchinov. Entretanto, na tradução disponível ao público brasileiro em 2001, a autoria de O freudismo é atribuída a Bakhtin, ainda que no Prefácio se faça menção à assinatura de Volóchinov, revelando a disputa pela autoria da obra. Neste trabalho, creditamos a Volóchinov a autoria dos dois textos sobre o freudismo, de modo a alinhar a autoria do artigo de 1925 à obra expandida de 1927. ABSTRACT Despite being rarely discussed in Bakhtinian Studies, Volóchinov’s critics towards Freudianism is a landmark in the theoretical production of Bakhtin’s Circle. Based on a critical analysis of Volóchinov’s position towards the therapeutic conditions of psychoanalysis, this article aims to investigate the psychoanalytic social event, in its therapeutic specificity, as a speech genre. To that end, we see social voice intertwining as a constitutive phenomenon of such an event. We reviewed the literature on the speech genres assumptions made by Bakhtin and his Circle to investigate the role of the setting in Freudian psychoanalytic therapy. Such role was then outlined through the Bakhtin’s Circle concepts of architectonics and heterodiscourse and Freud’s concepts of resistance and transference. We argue that the psychoanalytic discursive relationship is specific when it comes to the possibility of making the discursive interaction itself a space- time that is receptive to the unfinished dialogue, typical of both therapeutic encounter and elaborative speech. Keywords: verbal interaction; speech genres; heterodiscourse; psychoanalytic session. 2 A sessão de psicanálise como gênero do discurso 1. Palavras iniciais Nos escritos de Bakhtin e o Círculo4, um importante evento marca o percurso de um dos autores: o diálogo com a psicanálise. Em 1925, Volóchinov escreve o artigo Do outro lado do social: sobre o freudis- mo, expandido em 1927 na obra O freudismo: um esboço crítico. Em ambos os trabalhos, Volóchinov5 critica o método e os pressupostos teórico-filosóficos da psicanálise freudiana, entendida por ele como mais uma dentre as correntes teóricas subjetivistas e psicologizantes. Segundo Grillo (2017, p. 65), Volóchinov propunha “alternativas mais substanciais aos conceitos freudianos, por meio da valorização da pa- lavra ou da linguagem”, apresentando uma interpretação sociológica e discursiva para as relações entre discurso, consciência e atividade humana na sessão de psicanálise. Dado o teor discursivo dessas pro- postas, Moura-Vieira (2016, p. 58) afirma que os textos dedicados ao freudismo representam “um dos pilares da constituição do pensamento dialógico”, prenunciando o método sociológico de 1929. Trabalhos na área dos estudos bakhtinianos se dedicaram ao estudo dos escritos de Volóchinov sobre a psicanálise, atestando a incidência do contexto ideológico soviético dos anos 1920 nas críticas registradas pelo autor (Bezerra, 2017; Grillo, 2017; Lima & Perini, 2009; Moura-Vieira, 2016). Para Bezerra (2017, p. XIII), o contexto acadêmico-institucional em que O freudismo se insere estava fortemente marcado pelo enfoque 3 Eduardo da Silva Moll, Maria da Glória Corrêa di Fanti, Kelli Machado da Rosa marxista; por isso, Volóchinov estaria “tentando tensamente firmar posição no debate ideológico da época”. A título de exemplificação da demarcação de posições teórico-políticas na obra citada, ressaltamos o seu último capítulo, em que se avalia a relação entre marxismo e freudismo a partir da resposta a outros autores marxistas, tais como Bikhovski, Luria, Fridman e Zalkind, os quais atestavam a produtivi- dade do encontro de tais perspectivas teóricas. Volóchinov (2017, p. 109), em seu balanço final, defende que os “apologistas marxistas do freudismo” empreendiam a tarefa de “unir o impossível”, dado que, extraindo ou focalizando aspectos psicanalíticos e não outros, obscu- reciam aspectos nodais de ambas as perspectivas. Pela abordagem dialógica da linguagem, todo discurso citante é interessado. Dito diferentemente, ao citarmos o discurso dos outros, dele nos apropriamos responsivamente, demarcando posições autorais. Logo, O freudismo pode ser contemplado como um registro situado de uma apropriação singular, reelaborada e autoral das primeiras formulações de Freud pelo viés marxista, ideológico e discursivo. Como notam Lima e Perini (2009, p. 1. Palavras iniciais 81), tal apropriação opera com redutivismos relativamente à psicanálise, fazendo despontar, principal- mente, uma “identidade teórica” respondente à psicanálise, alocando- -a como subjetivista e alijada da história no próprio movimento de resposta. Na visão de Grillo (2017, p. 62), “Freud não é indiferente à influência das forças culturais e econômicas sobre a constituição da sociedade e do indivíduo”, muito embora o esforço argumentativo de Volóchinov defenda o contrário. Então, os pesquisadores bakhtinianos parecem concordar que O freudismo ganha relevância ao registrar a resposta a Freud – maturando, como defendem Moura-Vieira (2016) e Grillo (2017), o método sociológico concretizado em 1929 –, mas não como porta de entrada à teoria, à metodologia e à clínica psicanalíticas. Notamos que, em nosso campo, as relevantes leituras singulares efetuadas, observáveis nos trabalhos publicados, voltam-se mais à ponderação das críticas registradas na obra do que à compreensão de Volóchinov sobre o cerne da sessão de psicanálise, foco da nossa refle- xão neste artigo. Moura-Vieira (2016, p. 66) observa a produtividade do diálogo entre as ideias bakhtinianas e as psicanalíticas, indicando A sessão de psicanálise como gênero do discurso que esse diálogo, para ser fiel às ideias de Bakhtin e o Círculo, deve se inscrever “nos gêneros do discurso e da atividade humana que estão postos e se desenvolvem no horizonte social ampliado, estabilizando modos diversificados de operar a consciência humana na afirmação cotidiana do ato responsável e singular do sujeito dialógico”. Como veremos na sequência, o enfoque para a atividade humana nos gêneros do discurso já estava sendo lançado em O freudismo e será por nós retomado na condução do presente estudo. No texto de 1925, Volóchinov (2019, p. 81) não deixa de constatar que a psicanálise obteve “sucesso prático no campo do tratamento das neuroses”. Na obra de 1927, as condições para tal sucesso prático pa- recem encontrar respaldo na noção de “pequeno acontecimento social” psicanalítico, desenvolvido com base na dinâmica enunciativa engen- drada entre analista e analisando: “Nessas enunciações verbalizadas, reflete-se não a dinâmica da alma individual, mas a dinâmica social das inter-relações do médico com o paciente” (Volóchinov, 2017, p. 80). Grillo (2017, pp. 64 - 65) compreende, a partir dessas anotações, que “a sessão de psicanálise é um pequeno ato social produtor de enuncia- dos e signos ideológicos que só podem ser compreendidos na relação com os participantes sociais mais próximos, a situação social mais próxima e o horizonte social amplo”. 1. Palavras iniciais Assim, percebemos uma possível relação entre gênero do discurso, enunciado e terapia como elemento norteador do ato social psicanalítico pelas lentes de Volóchinov, dado que o autor enfoca as relações discursivas, responsivas e atitudinais entre o par analítico como cerne dessa prática terapêutica. Esse será o fio condutor da reflexão que aqui se inicia. Neste artigo, consideramos o atestado “sucesso prático” freudiano como um fenômeno sociológico-discursivo engendrado na sessão de psicanálise e temos como objetivo investigar o pequeno acontecimento social psicanalítico como um gênero do discurso em sua especificidade terapêutica, considerando o entrecruzamento de vozes constitutivo desse evento. Para tanto, empreendemos uma pesquisa de nível teórico, firmada em solo bakhtiniano, que dialoga com a teoria psicanalítica ao prospectar elementos que possivelmente se atualizem no evento real e concreto da atividade psicanalítica, orientando os sujeitos à ressigni- ficação axiológica dos conteúdos vivenciais pouco ou não elaborados 5 39.3 2023 Eduardo da Silva Moll, Maria da Glória Corrêa di Fanti, Kelli Machado da Rosa na e pela troca enunciativa firmada entre analista e analisando.6 Nesse sentido, entendemos “terapia” como “transformação” (inter)subjetiva na e pela linguagem, em que o uso de “na/pela” alude a aspectos rela- tivamente estáveis do gênero discursivo em questão, que podem vir a se concretizar entre sujeitos em terapia (Moll, 2022). Segundo Nunes (2018, p. 88), um dos elementos que caracteri- zam a interação na “sessão de psicanálise” é o setting, ou enquadre: é aquilo que “[...] dá forma à terapia, o que a situa no tempo e no espaço, através de um conjunto de determinações relativas, por exemplo, ao lugar em que a terapia ocorre, ao horário da sessão, aos objetos que compõem o consultório e até mesmo à postura do analista”. O enquadre indicia uma prática discursiva calcada no papel e na função do dizer e do ouvir alternadamente endereçados entre analista e analisando que, como veremos, conduz à terapia enquanto análise conjunta da psique. Todavia, somente o enquadre não garante o sucesso prático psicanalí- tico. Em nossa investigação bakhtiniana, devemos nos atentar, por um lado, à vivacidade e singularidade do evento de interação discursiva e, por outro, a seus elementos repetíveis, mais ou menos estáveis tanto do ato, quanto do gênero, como indica Sobral (2019); tal abordagem permite compreender o lugar do setting como condutor, mas não como definidor das práticas discursivas psicanalíticas. 6. Conforme a proposta deste artigo, nossa reflexão opera em nível teórico a partir dos textos do Círculo de Bakhtin e de Freud. Embora tenhamos ciência da importância e da contribuição de áreas que se dedicam à análise de atividades de trabalho, como a clínica da atividade (Clot & Faïta, 2000; Faïta, 2005) e a ergologia (Schwartz, 2016; Schwartz, Di Fanti & Barbosa, 2016), não recorreremos a essas abordagens devido ao fato de extrapola- rem o objetivo deste estudo. Entendemos, no entanto, que esta pesquisa apresenta potencial para desenvolver trabalhos futuros que contemplem gravações de sessões de psicanálise na concretude de seu acontecimento e/ou outras metodologias voltadas para o desenvolvi- mento de espaços de verbalização de experiências pessoais e coletivas (entrevistas, grupo de discussão etc.) em diálogo com áreas que se dedicam à análise da atividade profissional. 1. Palavras iniciais No cumprimento de nosso objetivo, recuperamos os pressupostos de Bakhtin e o Círculo que embasam o construto de gênero do discurso, assim como revisionamos o papel do enquadre na terapia psicanalítica freudiana. Conceitos como o de arquitetônica e de heterodiscurso para o Círculo, e o de resistência e de transferência para Freud respaldam nossa discussão. Seguimos as recomendações metodológicas de Voló- chinov (2018, pp. 220-221) de, ao analisarmos um enunciado, nunca 6 A sessão de psicanálise como gênero do discurso abstrairmos sua relação com o gênero discursivo e com a esfera da atividade em que ele se realiza, o que evidencia “a extrema importância do problema das formas do enunciado como uma totalidade”. Também, ancoramos nossa argumentação no mesmo esforço de Volóchinov em O freudismo, oferecendo uma leitura sociológico-discursiva às ideias psicanalíticas, prospectando, de nosso diálogo, caminhos para com- preender o fenômeno psicanalítico possivelmente atualizável na vida. Com isso em vista, nosso artigo se organiza da seguinte forma: (i) fazemos uma revisão bibliográfica do conceito de gêneros do discurso e dos demais conceitos a ele associados em Bakhtin, Medviédev e Vo- lóchinov; (ii) apresentamos uma breve incursão no conceito de setting para a prática psicanalítica, assim como nos conceitos de resistência, transferência e elaboração, lidos a partir do olhar bakhtiniano e, por fim, (iii) tecemos considerações acerca do enquadre e da análise como espaço-tempo aberto ao diálogo inconclusível. 2. Gêneros discursivos: o todo e a organicidade de suas partes Os conceitos de arquitetônica, de heterodiscurso e de enunciado se fazem relevantes no estudo dos gêneros discursivos, visto que res- paldam a alteridade, o tensionamento de vozes sociais e a atividade responsiva humana na e pela linguagem. Tendo em vista a complexi- dade desses conceitos, faremos recortes atinentes a obras específicas de Bakhtin e do Círculo, a partir dos quais empreenderemos nossa discussão. Em Para uma filosofia do ato responsável (1920-1924), Bakhtin (2017, p. 142) defende que o agir humano, permeado por valores sempre relacionais, concretiza um “princípio arquitetônico supremo do mundo real do ato”, a saber, “a contraposição concreta, arquitetonicamente válida, entre eu e outro”. De acordo com o autor, o ato é definido pelo contraste entre os polos do eu e do outro, centros de valores próprios da condição alteritária de existência humana, instaurando “caráter biplano da determinação valorativa do mundo” (Bakhtin, 2017, p. 142). Ainda, no ato, a valoração ou tom emotivo-volitivo é o resultado da partici- pação responsiva e expressiva do sujeito no existir, da “compreensão emotivo-volitiva do existir como evento na sua singularidade concreta, 7 39.3 2023 Eduardo da Silva Moll, Maria da Glória Corrêa di Fanti, Kelli Machado da Rosa sob a base do não-álibi do existir”, o não-álibi no ser (Bakhtin, 2017, p. 102). Ou seja, o ato em seu devir preserva a relação eu-outro como condição da resposta expressiva encarnada, valorada em relação ao outro e ao mundo cultural. Conforme Queiroz (2017), a consideração do agir em resposta ao outro e ao mundo social, situado num tempo e num espaço, torna possível “dizer que os gêneros do discurso atuam como formas con- cretas das diversas possibilidades arquitetônicas” do agir responsivo e responsável (pp. 637-638). Também segundo a autora, o princípio arquitetônico do ato acena à “tensão dialógica” própria das relações entre sujeitos, as quais “refletem e refratam discursos e tomam forma nas esferas da atividade humana” (Queiroz, 2017, p. 636). Logo, gêne- ro, enunciado e esfera se interpenetram conceitualmente no ensejo de conceber o agir alteritário humano: “para os princípios da abordagem dialógica, o encontro entre sujeitos e a consequente produção de gêne- ros discursivos só ocorre(rá) em dada esfera da comunicação social” (Barbosa & Di Fanti, 2020, p. 193). 2. Gêneros discursivos: o todo e a organicidade de suas partes A resposta não-indiferente ao outro, própria da arquitetônica do ato, mobiliza o conceito de voz alheia e de múltiplas vozes – hetero- discurso –, acenando à natureza alteritária do sujeito e do enunciado. Para Queiroz (2017, p. 636), nos momentos arquitetônicos eu-para-mim (como me vejo), eu-para-o-outro (como o outro me vê) e outro-para- -mim (como vejo o outro), ocorrem interações discursivas que “fazem ecoar vozes temporais e atemporais, no pequeno e no grande tempo, e são personificadas em um dado enunciado concreto, que tem função específica no mundo social, e são corporificadas num domínio cultural”. Essa multiplicidade de vozes remete à própria natureza da linguagem como heterodiscurso, conforme Bakhtin (2015, p. 67) postula em O discurso no romance (1934-1936): as línguas do heterodiscurso são “pontos de vista específicos sobre o mundo, formas de sua compreensão verbalizada, horizontes concreto-semânticos e axiológicos específicos”, fazendo reverberar apreciações, valorações e contestações diversas. A confluência de múltiplas vozes é, pois, a realidade discursiva concreta na e pela qual o sujeito age e se constitui; “o processo de formação ideológica do [sujeito] é um processo de assimilação seletiva das pala- vras dos outros” (Bakhtin, 2015, p. 135). Ou seja, na arquitetônica do ato concreto, as valorações do eu em resposta ao outro presentificam e 8 A sessão de psicanálise como gênero do discurso pessoalizam as variadas vozes constitutivas dos sujeitos e do contexto discursivo em que vivem. A concepção de enunciado relaciona-se à noção de arquitetônica, de heterodiscurso e de atividade humana quando enfocamos as esfe- ras ou os campos da criação ideológica. Em Marxismo e filosofia da linguagem (1929), Volóchinov (2018, p. 205, grifos do autor) defende que a palavra enunciada objetiva em seu interior o auditório social e situacional que a permeia – contexto plurivocal e pluriacentual he- terodiscursivo mais ou menos imediato – sendo um “ato bilateral”, determinado “tanto por aquele de quem ela procede, quanto por aquele para quem se dirige”, constituindo-se como “uma ponte que liga o eu ao outro”. A ponte entre os sujeitos é a palavra-ato que instaura, em sua arquitetônica alteritária, as recorrências e as singularidades do agir coletivo humano. Medviédev (2012, p. 7. As obras citadas pelas autoras são as mesmas utilizadas por nós e encontram-se nas referências deste artigo. 2. Gêneros discursivos: o todo e a organicidade de suas partes 190), em O método formal nos estudos lite- rários (1928), sustenta a ubiquidade da avaliação social – os sentidos opinativos compartilhados num meio ideológico – em qualquer ato enunciativo, indicando que “A presença da palavra é apenas um apên- dice de outra presença”, da presença do outro, dos valores comuns ao meio ideológico e à ação humana. Em Os gêneros do discurso (1952- 1953), Bakhtin (2016a, p. 57) afirma que todo enunciado “é pleno de ecos e ressonâncias de outros enunciados com os quais está ligado pela identidade da esfera da comunicação discursiva”. Compreendemos que o horizonte plurivocal e pluriacentual mais amplo permeia os senti- dos mais imediatos da arquitetônica do ato concreto e se inscreve nos enunciados, como notam Barbosa & Di Fanti (2020): É no enunciado, entendido por Bakhtin (2016) e Volóchinov (2018)7, como elo na cadeia da comunicação discursiva em sua relação indissociável com as esferas da atividade, que ecoam as ressonâncias de outros enunciados, tanto passados (via respostas) quanto futuros (via antecipações), o que configura distintas atitudes responsivas do locutor, não só em relação ao objeto do dizer, mas também acerca dos discursos outros sobre ele. (p. 189) 9 Eduardo da Silva Moll, Maria da Glória Corrêa di Fanti, Kelli Machado da Rosa Logo, o ato concreto, compreendido como enunciado, congrega em si as marcas valorativas da arquitetônica alteritária, reinstaurando o heterodiscurso constitutivo do agir responsável e, com isso, os ele- mentos repetíveis e irrepetíveis próprios das recorrências interativas que se ligam às esferas da atividade humana. A relação constitutiva entre esfera da atividade e inter-ação coletiva é o cerne do conceito de gênero para Medviédev (2012, p. 200): “o gênero é um conjunto de meios de orientação coletiva na realidade, dirigido para seu aca- bamento”. Volóchinov (2018, p. 94), por sua vez, entende que “Cada campo da criação ideológica possui seu próprio modo de se orientar na realidade, e a refrata a seu modo”. Ou seja, o construto de gênero do discurso põe em cena o ato responsivo dos sujeitos que, em relação, atualizam e reiteram sentidos mais e menos imediatos necessários à apreensão e à modificação da realidade. O princípio da responsividade do sujeito, o seu caráter singular e ativo em face ao outro e ao heterodiscurso, engendra o enunciado enquanto resposta a outros enunciados. Por isso, Bakhtin (2016a, p. 8. À época, a professora-pesquisadora Kelli Machado da Rosa assinava seus escritos como Kelli da Rosa Ribeiro. 2. Gêneros discursivos: o todo e a organicidade de suas partes 57) indica que “todo enunciado é repleto de variadas atitudes responsivas a outros enunciados de um dado campo da comunicação discursiva”. A alternância entre os sujeitos do discurso, em diálogo com o objeto já contestado pelas vozes alheias, gera o que Machado (2020, p. 157) chama de “circuito de respondibilidade”, em que “falante e ouvinte não são papéis fixados a priori, mas ações resultantes da própria mobiliza- ção discursiva no processo geral da enunciação”. Ou seja, dependendo da inter-ação, ligada a esferas distintas, os papéis assumidos pelos sujeitos se modificam e interferem na constituição dos enunciados e dos gêneros. Por isso, cada esfera discursiva “traduz uma atmosfera social de atividade, podendo se engendrar em diferentes contextos de interação verbal” (Ribeiro8, 2017, pp. 193-194): traduz modos típicos de direcionar-se ao outro e ao objeto ao agirmos no mundo. O lugar ocupado pelos sujeitos no circuito de respondibilidade influencia a constituição concreta do enunciado. Para Bakhtin (2016a), o todo temático, estilístico e composicional dos enunciados satisfaz tanto a individualidade dos falantes, que possuem projetos enuncia- 10 A sessão de psicanálise como gênero do discurso tivos singulares, quanto as recorrências do gênero, que apreendem a realidade à sua maneira. Como indicam Barbosa & Di Fanti (2020), a responsividade dos sujeitos engendra a totalidade do processo de formação dos enunciados a partir do projeto enunciativo, descrito por Bakhtin (2016a, p. 36) como “vontade de discurso do falante”. Assim, “podemos entender que, a partir de um projeto enunciativo, o locutor, na relação expressiva com o objeto do discurso e com o interlocutor, materializa o seu dizer em um gênero, o qual organiza o discurso via estilo, tema e construção composicional para a interação social” (Bar- bosa & Di Fanti, 2020, p. 190). O projeto de responder expressivamente ao outro faz do enunciado um ponto de encontro indissolúvel entre estilo, estrutura composicional e tema do(s) falante(s) e do gênero, na totalidade semântica de sua realização. Vale notar que a concepção de enunciado como resposta dá-nos um norte metodológico ao estudo dos gêneros do discurso. Como notam Barbosa & Di Fanti (2020), a vontade de responder ao outro qualifica o enunciado como ato expressivo, vivo e alteritário; logo, a interação responsiva atualiza elementos repetíveis e irrepetíveis dos gêneros. Estes, por sua vez, concretizam os discursos, os valores e as práticas nutridos pelas esferas de atividade, assim como nelas atuam, em relação de retroalimentação. 2. Gêneros discursivos: o todo e a organicidade de suas partes Assim, seguindo a ordem metodoló- gica de Volóchinov (2018), podemos pensar que o próprio enunciado traz as marcas orgânicas da complexa relação entre esfera e gênero, atualizando e pessoalizando em estilo, tema e estrutura composicional os projetos enunciativos dos sujeitos situados em interação. Sigamos, então, à caracterização desses constituintes orgânicos do enunciado, os quais refletem e refratam a interação responsiva mais e menos imediata. De acordo com Grillo (2020, p. 146), o tema “caracteriza-se por atribuir uma apreensão delimitadora do objeto do sentido e por compor- -se de uma expressão valorativa”. Ou seja, a depender das posições que os sujeitos ocupam em sua relação de alteridade, os valores e o grau de apreensão do objeto e da realidade irão compor diferentemente o tema da enunciação. O estilo é entendido como “uma atitude substancial e criadora do discurso com seu objeto, com o próprio falante e com a palavra do outro; ele procura fazer o material comungar organicamente com a linguagem e a linguagem com o material” (Bakhtin, 2015, p. 182). Em outros termos, são as escolhas linguístico-discursivas que 11 39.3 2023 Eduardo da Silva Moll, Maria da Glória Corrêa di Fanti, Kelli Machado da Rosa enformam o enunciado ao outro. A forma composicional corresponde a “formas relativamente estáveis e típicas de construção do conjunto” (Bakhtin, 2016a, p. 38, grifos do autor); esse conjunto é uma totalidade que instaura “vínculos composicionais” entre o projeto enunciativo e o enformamento do enunciado, que integram nossa “imaginação discursiva” (Bakhtin, 2016a, p. 43). Todos esses três elementos, tema, estilo e forma composicional, constroem a totalidade do enunciado, do ato e do gênero: “é o eco da totalidade do gênero que ecoa na palavra” (Bakhtin, 2016a, p. 53). Para Volóchinov (2018, p. 222), o conceito de gênero refere-se à ocorrência de “formas de comunicação cotidiana que sejam ao menos um pouco mais estáveis, fixadas pelo cotidiano e pelas circunstân- cias”. Essas formas ganham um “acabamento” de gênero, aquilo que corresponde “às particularidades ocasionais e singulares das situações cotidianas”, que dão forma relativamente estável à interação discursiva. Medviédev (2012, p. 198), em consonância com Volóchinov, argumen- ta que cada gênero “é um complexo sistema de meios e métodos de domínio consciente e de acabamento da realidade”. É válido destacar que, como nota Ribeiro (2017, p. 192), o acabamento relativo e a forma composicional do gênero estão ligados “a uma forma arquitetônica que dá contornos específicos aos enunciados”. 2. Gêneros discursivos: o todo e a organicidade de suas partes Não se trata, portanto, de um encaixotamento da realidade no e pelo gênero, mas da ação discursiva de enformar, de dar contorno discursivo, de criar sentidos na e pela linguagem a partir da disposição do eu e do outro numa dada esfera da atividade humana. Por fim, vale notar que os gêneros discursivos, uma vez experien- ciados, passam a compor a experiência cognoscente humana. Segundo Medviédev (2012, p. 196), cada gênero “é capaz de dominar somente determinados aspectos da realidade, ele possui certos princípios de seleção, determinadas formas de visão e de compreensão dessa reali- dade, certos graus na extensão de sua apreensão e na profundidade de penetração nela”. Pela ação ativa e responsiva do sujeito em seu meio ideológico, os gêneros passam a ser interiorizados, compondo a esfera interior de ativismo do sujeito: “a consciência humana possui uma série de gêneros interiores que servem para ver e compreender a realidade. Dependendo do meio ideológico, uma consciência é mais rica em gêneros, enquanto a outra é mais pobre” (Medviédev, 2012, p. 198). 12 A sessão de psicanálise como gênero do discurso Por isso, devemos também perceber a via de mão dupla entre gênero e consciência: os gêneros que experienciamos socialmente estruturam, de fora para dentro, a consciência responsiva dos sujeitos, assim como podem ser vividos ativamente na transformação da realidade objetiva. Em suma, compreendemos tema, estilo e forma composicional em ligação orgânica com as valorações, com a situação extraverbal, com a responsividade do sujeito e, principalmente, com a totalidade do ato enunciativo. Por isso, os gêneros, ao comporem nossa experiência cognoscente, orientam maneiras específicas de direcionamento do dito a um todo enunciativo, o qual é forjado no heterodiscurso em resposta a já ditos e na urgência a respostas vindouras. Esse todo forma-se a partir de projetos enunciativos dos sujeitos, os quais fazem reverberar em seus atos o trajeto formativo de sua consciência dialógica como apreensão seletiva das palavras alheias. Assim, uma postura de pesquisa atenta à totalidade do enunciado faz repercutir em cada uma de suas partes a arquitetônica, os centros de valores alteritários engendradores do ato, “a organização do sentido no todo englobante de um enunciado concreto que é realizado num ato responsável, enquanto potência no ser único e sempre em relação de interação com o outro, com a esfera ideológica, num determinado tempo-espaço” (Queiroz, 2017, p. 639). 2. Gêneros discursivos: o todo e a organicidade de suas partes A partir dessas ponderações, passemos a refletir sobre o enquadre no gênero sessão de psicanálise. 3. O setting no gênero sessão de psicanálise: experiência de encontro de vozes Em comunicações à sociedade leiga, Freud comumente relatava a especificidade da psicanálise em contraste com a psiquiatria e com as formas de tratamento mental da época. Em Compêndio de Psicanálise (1940), Freud (2018) avalia que “dentro e fora da ciência, basta supor que o psíquico é apenas a consciência” (p. 206). Sua novidade é o manejo clínico das “lacunas no psíquico, completando o que falta por meio de inferências plausíveis e traduzindo-o em material consciente”, produzindo “como que uma série complementar consciente para o psíquico inconsciente”, causador dessas lacunas (p. 208). Para tanto, tal contexto clínico precisaria de um elemento definidor: um discurso consciente que trouxesse à tona o inconsciente. A especificidade desse 13 Eduardo da Silva Moll, Maria da Glória Corrêa di Fanti, Kelli Machado da Rosa discurso é livre-associação. Haveria um pacto prévio entre analista e analisando, nomeado como “regra de ouro”, passo inicial e designativo da situação de análise, como lemos a seguir: Selamos um pacto. O Eu doente nos promete franqueza total, ou seja, que disporemos de todo o material que sua autopercepção lhe fornece; nós lhe garantimos rigorosa discrição e colocamos a seu serviço nossa experiência na interpretação do material influenciado pelo inconsciente. Nosso saber deve compensar sua insciência, deve restituir ao Eu o domínio sobre regi- ões perdidas da psique. Nesse pacto consiste a situação analítica. (Freud, 2018, p. 226) A livre-associação implica o analisando comunicar ao analista não apenas “o que diz intencionalmente e de bom grado, o que, como numa confissão, lhe traz alívio, mas também tudo o mais que sua auto- -observação lhe propicia, tudo o que lhe vem à mente, ainda quando lhe seja desagradável dizer, ainda quando lhe pareça irrelevante ou até mesmo absurdo” (Freud, 2018, p. 228, grifos do autor). A fala livre-associada nutre as possibilidades interpretativas e interventivas do analista, que deve se colocar em estado de atenção flutuante ao discurso do analisando. A atenção flutuante é, segundo Souza (2018, p. 74), a postura do analista que observa, interpreta e “recolhe o que existe de associativo na fala do sujeito no tratamento psicanalítico”. Ou seja, trata-se de um olhar global, não fixado em fragmentos discursivos específicos, capaz de conceber, no todo da fala e do agir do analisando, os traços associativos reveladores do abscôndito sintoma. Souza (2018, p. 3. O setting no gênero sessão de psicanálise: experiência de encontro de vozes 92) indica que na associação livre “o método psi- canalítico encontra não apenas o seu enquadre definidor, mas é através dela que institui sua dimensão ética, que a singulariza diante das outras formas de tratamento”. Para Celes (2005a, p. 29), tanto o princípio da livre-associação quanto a atenção flutuante do analista constituem os pilares da psicanálise como tratamento ao propiciar um trabalho conjunto de psico-análise (análise da psique): “no trabalho psicanálise trata-se de o analisando ‘falar’ o que foi esquecido, sob a condição do ‘ouvir’ específico do analista para que o analisando também ‘ouça’ o que fala”. Logo, o foco da prática psicanalítica “reside sempre no dis- curso do sujeito, ou melhor, nas potencialidades de sua associatividade fundamental” (Souza, 2018, p. 92). A fala livre associada, recebida em 14 A sessão de psicanálise como gênero do discurso atenção flutuante, monta o cenário mais imediato dos pontos de vista em embate, das vozes em diálogo e tensão na sessão. Em um viés bakhtiniano, poderíamos dizer que a fala livre asso- ciada, conforme pensada por Freud, indicia uma atividade humana que, na resposta ao analista, faz emergir no discurso temas e motivos que dificilmente seriam verbalizados em outras atividades. Haveria uma dificuldade social de verbalização de temas e motivos ideológi- cos específicos em outras esferas da comunicação discursiva que não aquela propiciada pelo contexto extraverbal psicanalítico. A sessão de psicanálise torna-se, então, um convite a falar o inaudito, com seus temas e conteúdos, organizando-os ideológica e enunciativamente para si e para o outro no e pelo engajamento discursivo do par analítico. A “livre associação”, nesse sentido, não pode ser pensada em ter- mos bakhtinianos como o “dizer livre”, ou o “dizer sem consequências”, dado que o enunciado sempre é objetivado por um sujeito responsável, constituído pelas relações alteritárias, sociais e ideológicas, sejam elas mais amplas ou mais imediatas. A “liberdade” do dizer pode ser pensada, dialogicamente, como o acolhimento responsivo que o psi- canalista destina à fala do analisando, para que este, ao endereçar seu dizer ao outro, também se escute. “Para a palavra (e consequentemente para o [ser]) não existe nada mais terrível do que a irresponsividade” (Bakhtin, 2016b, p. 105, grifos do autor); a presença responsiva do analista presta uma contrapalavra acolhedora àquilo que o sujeito não falaria em outras condições interacionais. 3. O setting no gênero sessão de psicanálise: experiência de encontro de vozes Daí a liberdade: a psicanálise, enquanto atividade, licencia projetos discursivos outros, para os quais estar falando corresponde estar sendo acolhido e, portanto, respondido pela presença ativa do analista. Seguindo a orientação metodológica de Volóchinov (2018), enten- demos que a relação orgânica entre esfera discursiva, gênero discursivo e enunciado engendra-se na atividade de dizer-se ao outro. Em outras palavras, na relação de alteridade entre analista e analisando, aquilo que em outras esferas discursivas poderia ser valorado pelo sujeito como execrável, insignificante ou disparatado, é acolhido como material da atividade psicanalítica. Assim, a troca enunciativa psicanalítica é um microcosmo da complexa interação entre valores, esferas discursivas e enunciados próprios da constituição dos sujeitos em diálogo, emer- 15 Eduardo da Silva Moll, Maria da Glória Corrêa di Fanti, Kelli Machado da Rosa gindo e dialogizando-se no enunciado livre-associado que se endereça ao analista. Portanto, o próprio gênero discursivo sessão de psicanálise comporta e anima projetos enunciativos que, em outros gêneros, per- maneceriam “censurados”, “não-oficiais”, inadequados, promovendo movimentos alteritários de fala e de escuta que socializam o conteúdo do psiquismo na pequena atmosfera social engendrada na atividade de análise. Como nota Peron (2004), a experiência de análise reflete a noção de cura não só como a possibilidade de “pensar aquilo que não pôde ser pensado” (p. 42), mas também como “um trabalho psíquico do paciente que o leve à aceitação de novos conteúdos: a elaboração” (p. 47)9. O conteúdo novo é tanto aquilo que se descobre no e pelo falar que se dirige ao analista, assim como é a resistência a essa descoberta: resiste-se ao que é encontrado quando se fala o “absurdo”, o “irrele- vante” ou o “desagradável”; entretanto, nas condições que permeiam a atividade de falar ao analista, reside a possibilidade de se envolver com a resistência e ressignificá-la ao direcioná-la a alguém. Em termos bakhtinianos, diríamos que a elaboração é o trabalho conjunto de falar e ouvir, que vai dando corpo discursivo às resistências do lembrar e do dizer, expandindo as possibilidades de dar contorno verbal àquilo que, fora da análise, permaneceria silenciado ou viria à tona com grandes percalços. Numa leitura teórica e sociológico-discursiva, poderíamos dizer que o pacto livre-associativo freudiano orienta o par analítico a ocupar posições arquitetônicas próprias da sessão de psicanálise. 9. O funcionamento da elaboração no trabalho psicanalítico, atinente ao tempo destinado ao analisando “para que ele se enfronhe na resistência agora conhecida, para que a elabore, para que a supere, prosseguindo o trabalho apesar dela, conforme a regra fundamental da análise”, encontra-se mais bem desenvolvido no texto Recordar, repetir e elaborar, de 1914 (Freud, 2010, p. 207-209). No entendimento de Jorge (2005, p. 60), a elaboração implica também a verbalização de tal processo: “a elaboração (Verarbeitung) confunde-se com o próprio trabalho analítico, na medida em que este visa essencialmente [à] simbolização”. 3. O setting no gênero sessão de psicanálise: experiência de encontro de vozes O enunciado concretiza a relação entre o eu e outro, trazendo em si as marcas da troca responsiva, as quais advêm dos novos polos axiológicos vivenciados na atividade psicanalítica. Nesses polos, não deveria haver julgamentos morais prévios e silenciadores entre o eu e o outro, mas sim um convite a elaborar, no discurso, as resistências e os silêncios. Nesse processo, inclusive os julgamentos do analisando-para-si poderiam vir a ser ex- 16 A sessão de psicanálise como gênero do discurso pressos, verbalizados ao outro, contribuindo para a socialização dos tons emotivo-volitivos que compõem a lembrança de sua história – a qual, por sua vez, é prenhe de palavras alheias e de vozes heterodiscursivas. Entendemos que o acolhimento promovido pela atividade analítica não seja neutro ou isento das visões de mundo do par analítico, mas tais visões de mundo, uma vez amparadas na técnica analítica, orientam- -se à atividade de análise e forjam um ambiente enunciativo no qual inclusive os julgamentos ao dizer de si podem ser dialogizados no enfrentamento às resistências implicadas nesse processo. Como observa Volóchinov (2017, p. 80), a resistência de que trata Freud “é, acima de tudo, uma resistência ao médico, ao ouvinte, em geral, à outra pessoa”: trata-se de uma postura responsiva ao novo – muitas vezes amedrontador e inusitado – da relação de análise. Poder enformar o dizer antes silenciado traz em si a descoberta e a resistência ao novo espaço elaborativo, sendo grande parte do trabalho analítico freudiano a exploração dessa resistência, dando-lhe novos sentidos e destinos verbalizados, gestualizados, entonados em algum material semiótico objetivado entre o par analítico. Pondo Bakhtin e Freud em diálogo na criação de inteligibilida- de sobre a atividade psicanalítica, entendemos que dizer o sintoma, vivenciá-lo com o analista, especifica a atividade psicanalítica como espaço-tempo de elaboração de um conteúdo temático que não encontra o mesmo espaço de verbalização nas demais esferas e gêneros. Então, o dizer do analisando faz ressoar tanto as vozes não enformadas do heterodiscurso, as “não-oficiais”, quanto as vozes mais estabilizadas, mais “oficiais”, provenientes dos inúmeros eventos de inter-ação nos quais o dizer é coletivizado sem maiores problemas. O par analítico coletiviza entre si aquilo que precisaria ser silenciado, ou que fora si- lenciado em outras atividades, dialogizando tais vozes a partir de um novo auditório social e de um novo contexto situacional – a sessão de psicanálise. 3. O setting no gênero sessão de psicanálise: experiência de encontro de vozes Por isso, podemos entender o conteúdo novo forjado na interação entre analista e analisando como “coenunciação dialogizada criativa” (Moll, 2022, p. 114). Tendo em vista a atividade-trabalho de psicanálise (Celes, 2005b), entendemos que o projeto enunciativo do sujeito que busca auxílio a questões de saúde mental encontra na sessão psicanalítica uma orienta- ção a papéis interacionais de fala e de escuta que, em sua especificidade, 17 Eduardo da Silva Moll, Maria da Glória Corrêa di Fanti, Kelli Machado da Rosa promovem encaminhamento terapêutico pela socialização dialogada, amparada pela técnica psicanalítica, dos conteúdos do psiquismo do analisando. O circuito de respondibilidade engendrado permite que os enunciados do dizer de si sejam escutados, acolhidos e respondidos pelo analista, assim como escutados pelo próprio analisando, encorpando o enunciado com os valores desse novo contexto interacional. Perce- bemos, em consonância com Volóchinov (2017), que nesse circuito essencialmente dialógico abre-se um espaço e um tempo novos, pro- pícios a que o analisando dê “uma resposta verbalizada e um desfecho verbalizado àquilo que fora reprimido e isolado no seu psiquismo, agravando-o” (p. 31). Esse desfecho – enformamento, acabamento – é permeado pelas posições axiológicas renovadas na atividade de psico- -análise conjunta, quando as múltiplas vozes constitutivas dos sujeitos se dialogizam na interação responsiva do par analítico em terapia. Dito isso, não podemos esquecer as conceituações mais amplas de setting, as quais se referem às características procedimentais, nor- mativas e atitudinais dos encontros analíticos. Para Zimerman (1999), é relevante destacar as propriedades globais concernentes à logística prévia das sessões: O setting, comumente traduzido em nosso idioma como enquadre, pode ser conceituado como a soma de todos os procedimentos que organizam, nor- matizam e possibilitam o processo psicanalítico. Assim, ele resulta de uma conjunção de regras, atitudes e combinações, tanto as contidas no ‘contrato analítico’ como também aquelas que vão se definindo durante a evolução da análise, como os dias e horários das sessões, os honorários com a respectiva modalidade de pagamento, o plano de férias... (Zimerman, 1999, p. 301) Numa leitura discursiva, depreendemos que o enquadre tem também a funcionalidade de assegurar a uma sessão de psicanálise o cumprimento de sua funcionalidade terapêutica. Logo, o enquadre traz em si os elementos repetíveis, pré-evento de trabalho analítico conjun- to, os quais incidem nos temas esperados a uma sessão de psicanálise, nos limites e nas possibilidades de apreensão valorativa da realidade. 3. O setting no gênero sessão de psicanálise: experiência de encontro de vozes Igualmente, orienta o estilo discursivo, ao indicar o regime enunciativo de livre associação, no qual palavras possíveis de serem ditas pelo ana- lisando comungarão com a previsão de acolhimento e interpretação do material livre-associado pelo analista. Esses aspectos são perpassados pelas formas típicas de construção de conjunto do enunciado, as quais 18 A sessão de psicanálise como gênero do discurso atualizam as possibilidades arquitetônicas terapêuticas previstas pelo gênero, traduzindo a atmosfera da atividade psicanalítica. Também, devemos pensar o enquadre a partir da perspectiva for- mativa do sujeito que se propõe analista. Franco & Kupperman (2020) defendem que a apropriação e a (re)criação de um enquadre interno sejam constitutivos do processo formativo do sujeito que pratica a psi- canálise. Para os autores, a apropriação garante a consolidação de olhar teórico e clínico-metodológico pessoalizado, embora sempre amparado na técnica. Isso sustentaria a possibilidade de exercer psicanálise em menor dependência com as contingências do enquadre externo, como as exigências concretas do espaço clínico: “o analista porta consigo o enquadre, encarna o enquadre, e sustenta o processo psicanalítico ainda que este não esteja inscrito nos parâmetros formais de um tratamento psicanalítico clássico” (Franco & Kupermann, 2020, p. 64). Além disso, os autores indicam que a aprendizagem da psicanálise traz consigo a apropriação da totalidade dessa prática pelo analista: o sujeito interioriza não só a teoria [...] mas todo um enquadre, composto a partir de elementos imaginários e afetivos que ele recolhe em suas experi- ências de análise, de supervisão, de leitura, de circulação institucional e de atendimento clínico supervisionado; é essa interiorização, compondo todas essas experiências em um único complexo (um nó), que oferece ao clínico as condições de trabalho psicanalítico”. (Franco & Kupermann, 2020, p. 65) Um semelhante processo de interiorização do enquadre é ressal- tado por Moreira & Esteves (2012) pela perspectiva da estabilidade relacional do tratamento tanto para o analista, quanto para o analisando. Em consonância com Franco & Kuppermann (2020), as autoras citadas indicam que o setting representa para o analista “um lugar para onde voltar”: nesse lugar estão as crenças, o aparato teórico-metodológico e a ética da prática. Para o analisando, o “espaço mental do setting” prolonga a “aliança terapêutica” para além do tempo e do espaço (Mo- reira & Esteves, 2012, p. 6). 3. O setting no gênero sessão de psicanálise: experiência de encontro de vozes Em sintonia teórica, Migliavacca (2008) concebe a apropriação do setting em estreita relação com a finalidade terapêutica da análise: O setting contempla arranjos práticos para a realização do trabalho, mas é também um conceito psicológico que inclui uma visão do que acontece dentro dele – da moldura – de modo diferente do que acontece fora. A par 19 Eduardo da Silva Moll, Maria da Glória Corrêa di Fanti, Kelli Machado da Rosa disso, mas não de menor importância, o setting se constitui como um objeto internalizado, estreitamente ligado ao vértice e à função analítica. O esclare- cimento necessário dos arranjos práticos é um dos pilares da moldura dentro da qual se desenhará em infinitas direções, o encontro de duas mentes, a do profissional e a de seu paciente, em busca de realização. (Migliavacca, 2008, p. 222) Em nossa leitura, a expansibilidade espaço-temporal do setting tornado signo interiorizado remete às experiências de análise prévias e acena aos gêneros interiores de que falava Medviédev (2012). Tor- nados parte da experiência cognoscente, esses gêneros trazem em si uma espaço-temporalidade arquitetônica engendrada pelos movimentos alteritários do evento analítico situado e pelas múltiplas vozes sociais que ressoam na resposta expressiva do sujeito a seu(s) outro(s). Ao se interiorizar no analista, o setting possibilita que arranjos arquitetônicos psicanalíticos se manifestem em suas mais distintas contingências nas ocasiões vindouras, em resposta a vozes outras. Ao ser interiorizado pelo analisando, o setting dá-lhe autonomia na vida ao findarem as sessões de análise, visto que traz em si a memória experiencial de um espaço-tempo analítico convidativo à dialogização de vozes sociais. A voz analítica ecoante, assim, constitui o analisando, dando-lhe uma referência enunciativa e axiológica para a possibilidade de elaboração infinda dos temas e motivos que o perpassam. Por fim, cabe-nos investigar a relação entre o setting e outro ponto nodal da metapsicologia freudiana: a transferência, conceito que espe- cifica o vínculo entre analista e analisando. Esse conceito complexo pode ser explicado, de forma geral, como a condição relacional que se dá quando analista e analisando entram em situação de análise: o primeiro passará a atuar e vivenciar, nas projeções inconscientes à figura do analista, seu passado psíquico (Zimerman, 1999). 3. O setting no gênero sessão de psicanálise: experiência de encontro de vozes No didático texto Cinco lições da Psicanálise, de 1910, Freud explica: “A transferência ocorre espontaneamente em todas as relações humanas, assim como entre o paciente e o médico; é sempre veículo da influência terapêu- tica, e seu efeito é maior quanto menos se suspeita de sua existência” (Freud, 2013, p. 281). No trecho que segue, menciona-se o caso da transferência na psiconeurose, mas iremos discutir a relação entre a metáfora química apresentada e a transferência: 20 A sessão de psicanálise como gênero do discurso [...] aquela parte da vida emocional do paciente que ele não pode mais evo- car na lembrança é vivenciada novamente na sua relação com o médico, e apenas com esse reviver na ‘transferência’ ele é persuadido da existência e do poder de tais impulsos sexuais inconscientes. Os sintomas, que, para recorrer a uma imagem da química, são precipitados de anteriores vivências amorosas (no mais amplo sentido), podem ser dissolvidos e transformados em outros produtos psíquicos apenas na elevação da temperatura da trans- ferência. (Freud, 2013, p. 280) [...] aquela parte da vida emocional do paciente que ele não pode mais evo- car na lembrança é vivenciada novamente na sua relação com o médico, e apenas com esse reviver na ‘transferência’ ele é persuadido da existência e do poder de tais impulsos sexuais inconscientes. Os sintomas, que, para recorrer a uma imagem da química, são precipitados de anteriores vivências amorosas (no mais amplo sentido), podem ser dissolvidos e transformados em outros produtos psíquicos apenas na elevação da temperatura da trans- ferência. (Freud, 2013, p. 280) Numa leitura sociológico-discursiva, diríamos que a transferência remete à vivificação responsiva dos temas, dos já ditos e dos não ditos que fazem parte da história do analisando ante a possibilidade de (re) endereçamento da palavra ao analista na relação de análise. Podemos compreender a transferência como uma relação de/em análise que tem, ela mesma, um potencial terapêutico no processo vivencial analítico, na posição responsiva ativa frente ao circuito de respondibilidade convidativo à fala em livre associação. Seguindo a metáfora catalíti- ca freudiana, podemos dizer que a valoração receptiva e acolhedora ao discurso livre-associado do analisando “eleva a temperatura” da relação e dissolve, pelo calor da escuta e da fala que se fez ouvida, a pedra sólida das vozes ansiosas em serem verbalizadas e endereçadas, portanto, conflitivas, porque ainda não elaboradas. 3. O setting no gênero sessão de psicanálise: experiência de encontro de vozes A transferência, como indica Peron (2004), enfoca o peso clínico da própria vivência relacional de análise; isso vai ao encontro de nossa discussão de en- quadre como experiência e também como memória de uma vivência interiorizada que vivificou o projeto enunciativo de ouvir e de ser ouvido para transformar-se. Ademais, vemos aqui uma relação discursiva entre o papel do enquadre, da resistência, da transferência e da elaboração nesse gênero em face às demais esferas da atividade humana. Na esfera da saúde mental, esses processos do gênero relacionam a sessão de psicanálise com demais esferas em função das vozes sociais que, no meio hete- rodiscursivo dialogizado, perpassam o analisando ao serem postas no jogo elaborativo. As vozes já enformadas dialogizam-se com as vozes em processo de dizerem-se e nisso se manifesta a resistência a dizer o abscôndito do sintoma, a qual só se dissolve na profundidade vincular entre o par analítico e no cronotopo (tempo-espaço) de análise. Pensan- do na relação orgânica entre enunciado, gênero e esfera (Volóchinov, 21 Eduardo da Silva Moll, Maria da Glória Corrêa di Fanti, Kelli Machado da Rosa 2018), a verbalização traz em si as marcas semântico-axiológicas da experiência de análise, fazendo a ampla esfera da saúde mental, na qual a atividade psicanalítica opera, altamente receptiva às demais vozes heterodiscursivas que constituem o sujeito, permeável às esferas outras, próprias do contexto discursivo globalizante dos analisandos. Portanto, acreditamos que a permeabilidade da esfera da saúde mental às demais esferas também especifica a atividade de elaboração tera- pêutica. Elaboração e análise somente podem ser pensadas na e pela relação do par analítico, a qual concretiza nos enunciados verbaliza- dos uma resposta outra ao inaudito, seja ele reflexo da resistência do analisando ao analista ou refração das experiências silenciadas nos demais gêneros e esferas. 4. Considerações finais No começo deste artigo, fizemos do interesse de Volóchinov pela psicanálise o nosso foco investigativo. Observamos as críticas que o integrante do Círculo de Bakhtin faz a aspectos teóricos, metodo- lógicos e filosóficos da psicanálise, mas não deixamos de notar as brechas para o desenvolvimento de uma pesquisa inspirada no êxito terapêutico desse pequeno acontecimento social. Assim, elencamos como objetivo investigar teoricamente o pequeno acontecimento social psicanalítico como um gênero do discurso em sua especificidade tera- pêutica, considerando o entrecruzamento de vozes constitutivo desse evento. Em sua leitura, Volóchinov (2017) destaca o peso da prática vivencial entre analista e analisando, a troca verbalizada, como vetor terapêutico. Neste artigo, não observamos uma prática concreta, mas observamos caminhos teóricos para pensar a dinâmica terapêutica em termos bakhtinianos. Então, perguntamo-nos sobre os aspectos que especificariam a orientação terapêutica dessa atividade e encon- tramos no enquadre um elemento que organizaria a arquitetônica do gênero, comporia a história formativa do psicanalista e engendraria um circuito de respondibilidade em que resistências, transferências e elaborações se tornariam possíveis. Assim, a consciência povoada por vozes sociais daria ao projeto enunciativo de curar-se contornos discursivos ao sintoma quando engajada na resposta ao psicanalista, em análise. 22 Agradecimentos O presente trabalho foi realizado com apoio do Conselho Nacional de Desenvolvimento Científico e Tecnológico – Brasil (CNPq) – Processos 130711/2020-9 e 311462/2020-0. A sessão de psicanálise como gênero do discurso 23 39.3 2023 Eduardo da Silva Moll, Maria da Glória Corrêa di Fanti, Kelli Machado da Rosa Eduardo da Silva Moll, Maria da Glória Corrêa di Fanti, Kelli Machado da Rosa Contribuição dos autores Nós, Eduardo da Silva Moll, Maria da Glória Corrêa di Fanti e Kelli Ma- chado da Rosa, declaramos, para os devidos fins, que não temos qualquer conflito de interesse, em potencial, neste estudo. Todos nós participamos da conceptualização do estudo, do desenho metodológico, da discussão do material de análise e da revisão do manuscrito. Todos os autores aprovam a versão final do manuscrito e são responsáveis por todos os aspectos, incluindo a garantia de sua veracidade e integridade. Conflito de interesses Declaramos não ter qualquer conflito de interesse, em potencial, neste estudo. A sessão de psicanálise como gênero do discurso A sessão de psicanálise como gênero do discurso Acreditamos ter encontrado na atividade do sujeito e no circuito de respondibilidade uma leitura possível para a especificação terapêutica desse gênero. Defendemos que a relação entre analista e analisando é específica no que tange à possibilidade de fazer da interação discursiva um espaço-tempo de encontro mais receptivo à palavra do outro. Tal ponderação remete à questão da alteridade, conforme reflete Di Fanti (2020), que implica a necessidade imperativa do outro – do seu olhar exotópico e do seu tom emotivo-volitivo – para a percepção de si próprio. É, nessa perspectiva, na arquitetônica valorativa concreta da sessão de psicanálise, que, seguindo Bakhtin (2003, p. 341), podemos entender a importância da verbalização para o processo de cura: “eu tomo consciência de mim e me torno eu mesmo unicamente me reve- lando para o outro, através do outro e com o auxílio do outro”. Nesse encontro com o outro, a palavra em elaboração apresenta- -se em caráter de coenunciação não-premeditada no espaço-tempo analítico. Em relação ao espaço-tempo de encontro, referimo-nos tanto ao enquadre interno formativo do analista (Franco & Kuppermann, 2020), fruto de encontros teóricos e clínicos passados, quanto à inte- riorização do enquadre (Moreira & Esteves, 2012; Migliavacca, 2008) pelo analisando, que dialogiza as múltiplas vozes constitutivas de si e experimenta, na relação com o outro, novos valores ao enformar sua história. O caráter da palavra não-premeditada acena à elasticidade analítica do enquadre em relação às demandas contingentes e singulares dos participantes, ao regime estilístico livre-associativo, assim como ao “diálogo inconclusível” de que trata Bakhtin (2003, p. 348), que não permite o fechamento do pensamento e da vida na relação com o outro. Com isso, entendemos que o enformamento enunciativo terapêutico não esgota o sintoma, mas faz emergir o ainda não dito, pensado ou experienciado a respeito da vida do analisando. Por fim, nossa visão de terapia como coenunciação é remetida às noções de elaboração e de transferência. Na coenunciação, vivências podem vir a ser (re) construídas nas condições alteritárias especificadas no gênero, que se vivificam na troca enunciativa. Igualmente, a potência criativa do diá- logo inconclusível instaura um espaço tenso de construção infinda do sujeito que se coloca ao olhar do outro para transformar-se em terapia. Referências Bakhtin, M. (2003). Reformulação do livro sobre Dostoiévski. In M. Bakhtin, Estética da criação verbal. Trad. de Paulo Bezerra. 4. ed. Martins Fontes. Bakhtin, M. (2015). Teoria do Romance I: A estilística. Trad. Paulo Bezerra. Editora 34. Bakhtin, M. (2016a). Os gêneros do discurso. In M. Bakhtin, Os gêneros do discurso. Trad. Paulo Bezerra. Editora 34.i Bakhtin, M. (2016b). O texto na linguística, na filologia e em outras ciências humanas. In M. Bakhtin, Os gêneros do discurso. Trad. Paulo Bezerra. Editora 34.ii Bakhtin, M. (2017). Para uma filosofia do ato responsável. Trad. Valdemir Miotello e Carlos Alberto Faraco. Pedro & João Editores. Barbosa, V. F., & Di Fanti, M. G. C. (2020). Notas sobre gêneros do discurso em Bakhtin, Volóchinov e Medviédev. In D. Rocha, B. Deusdará, P. Arantes, & M. Pessôa (Eds.), Em discurso 4 – Pesquisar com gêneros discursivos: interpelando mídia e política. Cartolina. https://www.editoracartolina.com.br/em-discurso-04 24 A sessão de psicanálise como gênero do discurso Bezerra, P. (2017). Freud à luz de uma filosofia da linguagem. In V. Volóchinov, O freudismo: um esboço crítico. Trad. Paulo Bezerra. Perspectiva. Celes, L. A.(2005a). Psicanálise é trabalho de fazer falar, e fazer ouvir. Psychê, 9(16), 25-48. http://pepsic.bvsalud.org/scielo.php?script=sci_ arttext&pid=S1415-11382005000200003 Celes, L. A. (2005b). Psicanálise é o nome de um trabalho. Psicologia Clínica, 17(2), 157-171. https://doi.org/10.1590/S0103-56652005000200012. Clot, Y., & Faïta, D. (2000). Genres et styles en analyse du travail: concepts et méthodes. Travailler, Revigny-sur-Ormain, 4, 7-42. Di Fanti, M. G. C. (2020). Notas sobre a alteridade em Bakhtin. In C. Paschoal et al. (Eds.), Círculo de Bakhtin: alteridade, diálogo e dialética. Polifonia. https://issuu.com/editorapolifonia/docs/circulo_ de_bakhtin-_alteridade__dialogo_e_dialetic. i Faïta, D. (2005). Análise dialógica da atividade profissional. Tradução e organização de Maria da Glória Corrêa di Fanti, Maristela Botelho França e Marcos Antonio Moura Vieira. Express. Franco, W. A. C., & Kupperman, D. (2020). Um lugar para pensar: uma hipótese sobre o enquadre interno do psicanalista. Jornal de Psicanálise, 53(99), 59-74. http://pepsic.bvsalud.org/scielo. php?script=sci_arttext&pid=S0103-58352020000200005&lng=pt &nrm=iso. Freud, S. (2010). 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https://openalex.org/W2768616513	https://vbn.aau.dk/ws/files/267229519/document_24_.pdf	English	null	Topical zoledronic acid decreases micromotion induced bone resorption in a sheep arthroplasty model	BMC musculoskeletal disorders	2,017	cc-by	5,696	Topical zoledronic acid decreases micromotion induced bone resorption in a sheep arthroplasty model DOI (link to publication from Publisher): 10.1186/s12891-017-1802-z Citation for published version (APA): Jakobsen, T., Kold, S., Shiguetomi-Medina, J., Baas, J., Soballe, K., & Rahbek, O. (2017). Topical zoledronic acid decreases micromotion induced bone resorption in a sheep arthroplasty model. B M C Musculoskeletal Disorders, 18(1), Article 441. https://doi.org/10.1186/s12891-017-1802-z General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. Citation for published version (APA): Jakobsen, T., Kold, S., Shiguetomi-Medina, J., Baas, J., Soballe, K., & Rahbek, O. (2017). Topical zoledronic acid decreases micromotion induced bone resorption in a sheep arthroplasty model. B M C Musculoskeletal Disorders, 18(1), Article 441. https://doi.org/10.1186/s12891-017-1802-z Aalborg Universitet Aalborg Universitet Topical zoledronic acid decreases micromotion induced bone resorption in a sheep arthroplasty model Jakobsen, Thomas; Kold, Søren; Shiguetomi-Medina, Juan; Baas, Jorgen; Soballe, Kjeld; Rahbek, Ole Published in: B M C Musculoskeletal Disorders DOI (link to publication from Publisher): 10.1186/s12891-017-1802-z Creative Commons License CC BY 4.0 Publication date: 2017 Document Version Publisher's PDF, also known as Version of record Link to publication from Aalborg University Citation for published version (APA): Jakobsen, T., Kold, S., Shiguetomi-Medina, J., Baas, J., Soballe, K., & Rahbek, O. (2017). Topical zoledronic acid decreases micromotion induced bone resorption in a sheep arthroplasty model. 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Jakobsen et al. BMC Musculoskeletal Disorders (2017) 18:441 DOI 10.1186/s12891-017-1802-z Open Access © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. * Correspondence: Thomas.Jakobsen@ki.au.dk 1Orthopaedic Research Laboratory, Department of Orthopaedics, Aarhus University Hospital, Norrebrogade 44, Building 1A, DK-8000 Aarhus, Denmark 2Department of Orthopaedics, Aalborg University Hospital, Aalborg, Denmark Abstract Background: Initial micromotion of a total hip replacement is associated with aseptic loosening. The use of bisphosphonates could be one way to reduce peri-implant bone resorption induced by micromotion. Bisphosphonates compounds are inhibitors of bone resorption. The aim of this study was to investigate whether local treatment with bisphosphonate would reduce bone resorption and fibrous tissue around an experimental implant subjected to micromotion. Methods: One micromotion implant were inserted into each medial femoral condyle in ten sheep. During each gait cycle the implant axially piston 0.5 mm. During surgery one of the femoral condyles were locally treated with 0.8 mg zoledronate. The other condyle served as control. Observation period was 12 weeks. Results: Histological evaluation showed a fibrous capsule around both the control and bisphosphonate implants. Histomorphometrical analysis showed that 97% of the surface on both control and bisphosphonate implants were covered by fibrous tissue. However, the bisphosphonate was able to preserve bone in a 1 mm zone around the implants. Conclusion: This study indicates that local treatment with bisphosphonate cannot prevent the formation of a fibrous capsule around an implant subjected to micromotion, but bisphosphonate is able to reduce resorption of peri-prosthetic bone. Keywords: Bisphosphonate, Osseointegration, Implant, Animal, Arthroplasty implant failure [3]. Early bone resorption can be initiated by an unstable implant generating micro-movement or fluid pressure [6–8]. We have previously shown that implant micromotion without the presence of wear debris can generate a fibrous membrane and cause bone resorption [9]. In vivo studies suggest that instability induced bone resorption works through similar mo- lecular mechanisms as particle induced bone resorp- tion [10, 11]. Aseptic loosening of an implant involves several steps. One of the first steps could be bone resorption due to micro-movement of the implant. The resorption bone is replaced with fibrous tissue. Wear debris will subsequently migrate from the joint articulation along the fibrous tissue to the bone to implant interface and aggravate the process [12, 13]. Longevity of the implant might potentially be increased if micro- Topical zoledronic acid decreases micromotion induced bone resorption in a sheep arthroplasty model Thomas Jakobsen1,2*, Søren Kold1,2, Juan Shiguetomi-Medina1, Jorgen Baas1, Kjeld Soballe1 and Ole Rahbek1 Background Initial stable implant fixation is important for long-term survival of total joint replacements [1, 2]. Radiostereo- metrical studies indicate that early migration is a associated with later revision [1–3]. Treatment strategies that optimize initial implant fixation and prevents early implant migration could have the potential to reduce risks of later revision. Failure of initial implant osseointegration will result in early implant migration and bone resorption [4]. Early implant migration is associated with reduced resistance to withstand implant movement induced by external load [5]. Continues implant migration is associated with Jakobsen et al. BMC Musculoskeletal Disorders (2017) 18:441 Page 2 of 7 Page 2 of 7 movement of the implant were not allowed to induce bone resorption. Our sample size calculation was based on a previous study [9]. We assumed the standard deviation of the relative change to be 50%. Two-sided α and β were set to 5% and 20%, respectively. We added two extra ani- mals to the calculated sample size of eight to counteract if implants from one or two animals were lost for subse- quent analysis. p One way to reduce micromotion induced bone resorp- tion and fibrous membrane formation could be with the use of bisphosphonates. These compounds binds strongly to bone, inhibit osteoclastic activity, and reduce bone resorption [14]. Systemic bisphosphonate treatment has previously in an experimental model of osteolysis been able to reduce bone resorption [15]. We have previously shown that local treatment with bisphosphonate can in- crease new bone formation, preserve lamellar bone and allograft, and increase biomechanical implant fixation in an experimental joint replacement model [9]. In a clinical study, local treatment with bisphosphonate was shown to reduce acetabular component migration measured by radiostereometrical analysis [16]. The same effect has been observed in the study investigating local ibandronate treat- ment in total knee arthroplasties [17]. Others have shown the local or systemic treatment with bisphosphonates can reduce but not prevent osteolytic bone resorption and formation of fibrous tissue in a rat model of prosthetic loosening [18, 19]. Our group has previously shown in a large animal model that systemic alendronate can reduce bone resorption around unstable implants but not prevent it [20]. A stronger stimulus might be needed. Only way to obtain a stronger stimulus could be with topical adminis- tration of a third generation bisphosphonate. Background The aim of this study was to investigate in a sheep arthroplasty model whether topical treatment with bisphosphonate would reduce bone resorption and fibrous tissue. Study design We used 10 skeletally mature Danish Landrace sheep with a mean weight of 40 kg (range, 35–50 kg). The sheep were obtained though our Institutional farm (Påskehøjgaardcentret, 8380 Trige, Denmark) affiliated with our Research facility. Our Institutional Animal Care and Use Committee approved the study. Institutional guidelines for the treatment and care of experimental animals were followed. Implants We used 20 custom-made micromotion implant devices (Fig. 1). Our micromotion device has previously been described in detail [9, 21]. In short, the micromotion de- vice allows a polymethylmethacrylate (PMMA) implant to piston 0.5 mm with respect to the bone during each step the animal takes. The micromotion creates shear forces in the bone to implant interface. The implants in this study were made sterile by gamma irradiation (25– 50 kGy for 16 h, Codan Steritex, Espergaerde, Denmark). Surgery With the sheep under general anesthesia, we used our standard anteromedial surgical approach to the knee [9, 21, 22]. Our micromotion device was inserted into the weight-bearing portion of the medial femoral condyle. The procedure has been described in detail in our previously study [9]. During each surgery, we tested and assured that the micromotion device would piston during each gait cycle. Before the PMMA implant was mounted onto the an- chor screw, we soaked the bone cavity for 60 s with either saline as control or 1 mL saline containing 0.8 mg zole- dronate (Aclasta, Novartis Healthcare A/S, Copenhagen, Denmark) as intervention. After soaking the bone for 60 s, the cavity was rinsed with saline and excess zoledronate or saline together with blood coming from the marrow cavity was sucked away. In this study, all sheep were weight bearing on the operated leg within 5 days of surgery. After the 12 weeks observation period the sheep were euthanized with an overdose of hypersaturated barbiturate and the bones were collected for preparation and analyses. At harvest, all pistons were movable. We tested the hypothesis that local zoledronate treat- ment would reduce bone resorption and fibrous tissue formation around implants subjected to micromotion. Specimen preparation Bone specimens were stored at −20 °C immediately. We cut each specimen perpendicular to the long axis of the implant using a water-cooled bad saw (Exact Apparatebau, Nordenstedt, Germany) (Fig. 2). The specimens was fixed in 70% ethanol and used for later analysis. Preparation and subsequent evaluation were blinded. In this study, we used a paired design with intervention and control group in the same sheep. We have previously shown that the used model of micromotion is able to in- duce bone resorption [9]. In each sheep, we inserted one loaded micromotion device (Fig. 1) into each of the medial femoral condyles. Zoledronate was administered locally in one of the knees. Saline was used as control in the contra- lateral knee. The zoledronate and control implants were systematically alternated between left and right knee. Our observation period was 12 weeks. Surgery One sheep died during surgery and was excluded. The remaining 9 sheep completed the 12-weeks observation period. No clinical signs of infection were present at time of euthanization. We used a stereological software program (newCAST, Visiopharm A/S, Horsholm, Denmark) for the histomor- phometrical analysis. Volume fractions of bone were estimated by point counting [25] in a zone from the im- plant surface and 1 mm into the surrounding bone. Fractions of bone in contact with the implant surface were estimated using sine-weighted lines [26]. Histology and Histomorphometry All bone-implant specimen were dehydrated in ethanol (70%–96%) containing basic fuchsin, and embedded in Epoxy (EPOFIX, Struers, Copenhagen, Denmark). Four Page 3 of 7 Jakobsen et al. BMC Musculoskeletal Disorders (2017) 18:441 Fig. 1 Schematic drawing showing implant position in the medial femoral condyle. Reproduced with permission from copyright holder/author transformed and Student’s paired t-test was performed on absolute differences between normally distributed log-transformed paired data. An absolute difference between the logarithms of a pair of data equals the logarithm of the ratio within the pair [27]. 25-μm thick sections were cut with a hard tissue microtome (KDG-95, MeProTech, Heerhugowaard, The Netherlands) around the center part of each implant [23]. The sections were cut with a distance of 400 μm. Before making the sections, the implant was randomly rotated around its long axis. The sections were cut parallel to this axis. These techniques provide reliable results with negligible bias [24]. Before the sections were mounted on glass, we surface stained them with 2% light-green (BDH Laboratory Supplies, Poole, England). Histology The most striking difference between the control and zoledronate implants was the presence of a 200–300 μm thick fibrous membrane around the control implants (Fig. 3). A fibrous membrane was also present around the zoledronate implants, but the membrane was thinner with an approximately thickness of 50 μm. The fibrous membrane in both the control and zoledronate group consisted of dense paralleled fibers. No qualitative mor- phological difference was found between the two groups Statistical analysis Intercooled Stata 9.0 (Stata Inc., College Station, TX, USA) were used for statistical analysis. Statistical analyses were done on ratios between paired data, which were not normally distributed. All variables were log- Fig. 2 Schematic diagram showing the specimen preparation. Each bone-implant specimen is embedded and cut into four slides for histomorphometrical analysis. Reproduced with permission from copyright holder/author Fig. 2 Schematic diagram showing the specimen preparation. Each bone-implant specimen is embedded and cut into four slides for histomorphometrical analysis. Reproduced with permission from copyright holder/author Jakobsen et al. BMC Musculoskeletal Disorders (2017) 18:441 Page 4 of 7 Fig. 3 Representative photomicrographs of samples from the same animal. The samples were stained with basic fuchsin and counterstained with 2% light green. Note the thick fibrous membrane around the control implant compared to the zoledronate implant. Solid bar = 1 mm. Dotted bar = 0.3 mm Fig. 3 Representative photomicrographs of samples from the same animal. The samples were stained with basic fuchsin and counterstained with 2% light green. Note the thick fibrous membrane around the control implant compared to the zoledronate implant. Solid bar = 1 mm. Dotted bar = 0.3 mm (95% CI: 94% - 99%) in the control group (p = 0.65). We found no significant differences in the surface-fractions of woven, lamellar, and total bone (p = 0.76, p = 0.42, p = 0.71). when looking at the bone in the peri-implant zone. The bone consisted mainly of lamellae with spindle-shaped osteocytes in between. Sparse amount of osteoclasts were observed in both groups. No retained bone debris was observed in any of the groups. Discussion The purpose of this study was to investigate whether topical treatment with zoledronate could prevent bone resorption and fibrous tissue formation in our large animal model of micromotion induced bone resorption. We found that zoledronate did not prevent formation of a fibrous membrane, but was able to reduce bone re- sorption and thickness of the fibrous membrane. The local zoledronate treatment preserved 14% (p = 0.02) more total bone around the implants in a 1 mm zone in the zoledronate group compared to the control group (Fig. 4). We found a total peri-implant bone volume fraction of 57% (95% CI: 46% - 67%) in the control group and 66% (95% CI: 53% - 78%) in the zoledronate group. No statistically significant changes were found when comparing bone volume fractions for woven (p = 0.08) or lamellar (p = 0.32) bone (Fig. 3). In the zoledronate group, 16% (95% CI: 2% - 29%) of 1 mm peri-implant zone was made of fibrous tissue compared to 23% (95% CI: 14% - 33%) in the control group (p = 0.22). The used model is intended to imitate the bone to cement interface of a total hip replacement. We have previously demonstrated that our experimental model are able to induce peri-implant bone resorption and therefore also suitable to test local adjuvant therapies against bone resorption [9]. The paired design allowed us to eliminate the biological difference between individuals. The implant surfaces in the both the zoledronate and control group were virtually covered by fibrous tissue. The surface fraction for fibrous tissue was 97% (95% CI: 94%- 100%) in the zoledronate group compared to 97% Our study is limited by an observation period of 12 weeks and only nine animals completed the study. Jakobsen et al. BMC Musculoskeletal Disorders (2017) 18:441 Page 5 of 7 Fig. 4 Tissue-volume fractions in a 0–1 mm zone around implants. Paired data connected by line Non-significant differences should therefore be inter- preted with caution. An interesting histological finding is the difference in thickness of the fibrous membrane between the zoledro- nate and control groups. We know from previous stud- ies from our group that micromotion is able to induce formation of a fibrous membrane [9, 34]. In this study we observed that local zoledronate treatment histologi- cally reduced the thickness of the membrane. Discussion It could be that shear forces and stain at the tissue-to-implant inter- face are to high to by counteracted by zoledronate. Further away form the implant surface, strain and shear forces diminishes and zoledronate are able to partly preserve bone. We know from previous studies that in- creased amount of bone and decreased amount of fi- brous tissue in correlated to increased implant stability [22]. The difference in fibrous layer thickness should therefore have the potential to make the zoledronate implants more mechanically stable. Zoledronate is a potent inhibitor of bone resorption [28]. We have previously shown that local treatment with zoledronate can inhibit resorption of allograft and increase fixation of both primary and revision implants [29, 30]. In this study, we show that local treatment with zoledronate can reduce resorption of bone in a 1 mm peri-implant zone around an implant subjected to controlled micromotion. Our model of micromotion has a piston that allows a 0.5 mm movement of the implant during each loading. The model was designed to imitate a cemented femoral implant subjected to micromotion. The amplitude of movement of our implant is above the accepted thresh- old of 0.15 mm studies and suggested by Van der Voort based on RSA and thereby comparable to clinical implants with a high risk of as aseptic loosening [31]. Furthermore, the loading conditions of both clinical femoral implants and our experimental implants are comparable; both implants transfers load by shear forces though the bone-cement imterface. In our model, move- ment will occur at the implant-to-bone interface during each gait cycle and thereby create high shear forces and strain between the implant and tissue in contact with the implant. According to studies by and Carter and Giori, the amplitude of strain dictates which tissue can be formed [32, 33]. A high strain will induce bone re- sorption and formation of fibrous tissue. We found that local zoledronate treatment were able to partly counter- act the strain/micromotion induced bone resorption and preserve 14% more total bone compared to our control. It has previously been shown that systemic alendro- nate is able to inhibit bone resorption in a canine osteo- lytic hip arthroplasty model [15]. An animal study using a rodent model has shown that systemic treatment with alendronate or clodronate can reduce instability-induced bone resorption, but high doses are needed [19]. PE: polyethylene; PMMA: polymethylmethacrylate; RSA: Radiostereometric Analysis Acknowledgements h h h h The authors wish to thank Jane Pauli, Orthopaedic research Laboratory, Aarhus University Hospital, for technical expertise. The authors declare to have no professional or financial conflict of interest. The authors wish to thank Jane Pauli, Orthopaedic research Laboratory, Aarhus University Hospital, for technical expertise. The authors declare to have no professional or financial conflict of interest. Authors’contributions TJ, SK, JB, KS, and OR: Designed the study. TJ, SK, JB, and OR: performed the surgery. TJ and JSM: specimen preparation and histomorphometry. TJ, and JB: push-out test. TJ: Statistical analysis. TJ, SK, JSM, JB, KS, and OR: Interpretation of data. TJ wrote the first draft of the manuscript. SK, JSM, JB, KS, and OR: critically revision of manuscript. All authors read and approved the final manuscript. TJ, SK, JB, KS, and OR: Designed the study. TJ, SK, JB, and OR: performed the surgery. TJ and JSM: specimen preparation and histomorphometry. TJ, and JB: push-out test. TJ: Statistical analysis. TJ, SK, JSM, JB, KS, and OR: Interpretation of data. TJ wrote the first draft of the manuscript. SK, JSM, JB, KS, and OR: critically revision of manuscript. All authors read and approved the final manuscript. Local treatment with zoledronate was not able to pre- vent formation of peri-implant fibrous tissue. However, zoledronate was able to reduce the thickness of the peri- implant fibrous membrane. This is in agreement with results from another study where local alendronate treatment reduced formation of soft tissue, but did not prevent its formation [19]. It is also in agreement with a clinical study were local ibandronate treatment reduced occurrences of radiolucent lines around acetabular cups lines, but not prevented them [16]. Based on our previ- ous study were systemic alendronate reduced bone re- sorption around an unstable implant, we expected that a stronger anti-resorptive stimulus could be obtained with local zoledronate. We therefore expected that local zole- dronate could prevent bone resorption and formation of fibrous tissue. It may be that implant micromotion is a too strong stimulus for even local zoledronate being able to completely prevent bone resorption. Another explan- ation could be that the single dose of zoledronate acts only as a defense again bone-resorption for a limited amount of time. Zoledronate, released from bone by osteoclastic resorption, could be slowly washed away from the implant-bone interface. By time the concentra- tion will be too low to inhibit the continuous strong bone resorptive stimulus from micromotion. Funding g This study was unconditionally funded by The Danish Ministry of Higher Education and Science. This study was unconditionally funded by The Danish Ministry of Higher Education and Science. Discussion One likely explanation for the preserved bone volume density found in this study is the inhibitory effect of zoledronate on bone resorption. Previous studies have shown that preservation of lamellar bone often leads to increased formation of new bone [35–37]. The increased new bone formation is explained by increased osteoconductive properties of the preserved lamellar bone. In this study, we are not able to show a statistically significant increase in peri-implant formation of new bone (8% in Jakobsen et al. BMC Musculoskeletal Disorders (2017) 18:441 Page 6 of 7 Page 6 of 7 zoledronate group vs. 3% in control group, p = 0.08). One possible explanation could be a to short observation period for new bone formation to occur. The effect on bone formation and resorption has been shown to be different over time [37]. A longer observation period might have shown a more pronounced effect of zoledro- nate on both bone formation and resorption. References 1. Kärrholm J, Borssén B, Löwenhielm G, Snorrason F. Does early micromotion matter? 4-7 year stereoradiographic follow-up of 84 cemented prostheses. J Bone Jt Surg - Br Vol. 1994;76:912–7. 1. Kärrholm J, Borssén B, Löwenhielm G, Snorrason F. Does early micromotion matter? 4-7 year stereoradiographic follow-up of 84 cemented prostheses. J Bone Jt Surg - Br Vol. 1994;76:912–7. In clinical practice, soaking the bone bed before implantation is a simple procedure. 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Submit your next manuscript to BioMed Central and we will help you at every step: Submit your next manuscript to BioMed Central and we will help you at every step: Submit your next manuscript to BioMed Central and we will help you at every step: • We accept pre-submission inquiries • Our selector tool helps you to ﬁnd the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit and we will help you at every step: 34. Bechtold JE, Mouzin O, Kidder L, Part II. Implementation with loaded titanium implants and bone graft. Acta Orthop Scand. 2001;72:650–6. 35. Baas J, Elmengaard B, Jensen TB, Jakobsen T, Andersen NT, Soballe K. The effect of pretreating morselized allograft bone with rhBMP-2 and/or pamidronate on the fixation of porous Ti and HA-coated implants. 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https://openalex.org/W3036667400	https://digitalcollection.zhaw.ch/bitstream/11475/20208/3/2020_Grylka_etal_Evaluation_BMC-Health-services.pdf	English	null	Evaluation of a midwifery network to guarantee outpatient postpartum care: a mixed methods study	BMC health services research	2,020	cc-by	9,673	Grylka-Baeschlin et al. BMC Health Services Research (2020) 20:565 https://doi.org/10.1186/s12913-020-05359-3 Grylka-Baeschlin et al. BMC Health Services Research (2020) 20:565 https://doi.org/10.1186/s12913-020-05359-3 Open Access Evaluation of a midwifery network to guarantee outpatient postpartum care: a mixed methods study sanne Grylka-Baeschlin1* , Carolina Iglesias2, Rebekka Erdin1,2 and Jessica Pehlke-Milde1 Susanne Grylka-Baeschlin1* , Carolina Iglesias2, Rebekka Erdin1,2 and Jessica Pehlke-Milde1 Correspondence: Susanne.Grylka@zhaw.ch 1Research Unit for Midwifery Science, ZHAW Zurich University of Applied Sciences, Technikumstr. 81, CH-8401 Winterthur, Switzerland Full list of author information is available at the end of the article Abstract Background: The necessity of outpatient postpartum care has increased due to shorter hospital stays. In a health care system, where postpartum care after hospital discharge must be arranged by families themselves, this can be challenging for those experiencing psychosocial disadvantages. Therefore, we compared characteristics of users of a midwifery network which referred women to outpatient postpartum care providers with those of women organising care themselves. Additionally, we investigated benefits of the network for women and health professionals. Methods: Evaluation of the services of a midwifery network in Switzerland. We combined quantitative secondary analysis of routine data of independent midwives with qualitative telephone interviews with users and a focus group with midwives and nurses. Descriptive statistics and logistic regression modelling were done using Stata 13. Content analysis was applied for qualitative data. Results: Users of the network were more likely to be: primiparas (OR 1.52, 95% CI [1.31–1.75, p < 0.001]); of foreign nationality (OR 2.36, 95% CI [2.04–2.73], p < 0.001); without professional education (OR 1.89, 95% CI [1.56–2.29] p < 0.001); unemployed (OR 1.28, 95% CI [1.09–1.51], p = 0.002) and have given birth by caesarean section (OR 1.38, 95% CI [1.20–1.59], p < 0.001) compared to women organising care themselves. Furthermore, users had cumulative risk factors for vulnerable transition into parenthood more often (≥three risk factors: 4.2% vs. 1.5%, p < 0.001). Women appreciate the services provided. The collaboration within the network facilitated work scheduling and the better use of resources for health professionals. Conclusions: The network enabled midwives and nurses to reach families who might have struggled to organise postpartum care themselves. It also facilitated the work organisation of health professionals. Networks therefore provide benefits for families and health professionals. Keywords: Postpartum care, Midwifery, Network, Psychosocially disadvantaged families Keywords: Postpartum care, Midwifery, Network, Psychosocially disadvantaged families Background The length of hospital stay after childbirth declined in Switzerland from 5.7 days in 2001 to 4.4 days in 2015 (Fed- eral statistical Office, Medical statistics of hospitals, unpub- lished data). As observed in other European countries, the implementation of Diagnosis Related Groups (DRG) for in- voicing health care costs led to increased pressure on hospi- tals to discharge patients earlier [20]. The reason for this is that case based flat rates are invoiced to health insurances in- stead of the effective costs. In Switzerland, out of hospital postpartum care consisting of home visits by midwives is provided until the 56th day after birth and is regulated by the Health Care Insurance Act [21]. At the same time as the decline of the length of postpartum hospital stay, the use of out of hospital postpartum care provided by self-employed midwives and, to a lesser extent, also self-employed nurses with work experience in postpartum care, increased [22]. The majority of families in Switzerland make use of postpar- tum care after hospital discharge [22, 23]. However, postpar- tum care following hospital discharge has to be organised by the families themselves. Women find contact details for mid- wives and nurses in the internet but often need to call several health professionals to be successful. In some Swiss regions and during holiday periods, women have difficulties finding a midwife. The observed shift toward out of hospital postpar- tum care therefore emphasises the need for well-organised care providers. In different Swiss areas, midwifery networks assume the organisation of guaranteed out of hospital post- partum care. One of them is called “Familystart Zurich” and provides its services in the Canton of Zurich [24]. Women using these services register online themselves or are regis- tered by the hospital and are mediated in an easy and un- complicated way to postpartum care providers. Antenatal care providers and hospitals promote these services. g In a health care system where out of hospital postpar- tum care has to be arranged by families themselves, this can be challenging for those experiencing psychosocial disadvantages. The first days after childbirth are a crit- ical phase in the lives of mothers and their children and young families need support to overcome fears related to the transition into parenthood [1–4]. A safe early dis- charge from hospital requires access to guaranteed post- partum care at home in order to avoid readmission to hospital [5, 6]. Aim of the study The aim of this study was to assess the benefits of guaran- teed postpartum care through a midwifery network for families, especially socially disadvantaged ones, and health professionals. We investigated: a) how users of the services of the network differed from women organising their post- partum care themselves, b) women’s satisfaction and ben- efits and c) midwives’ and nurses’ satisfaction and benefits due to the services of the network. © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Grylka-Baeschlin et al. BMC Health Services Research (2020) 20:565 Grylka-Baeschlin et al. BMC Health Services Research (2020) 20:565 Page 2 of 12 Grylka-Baeschlin et al. BMC Health Services Research (2020) 20:565 Background In some health care systems in Central Europe, postpartum care after hospital discharge has to be organised by the families themselves [4, 7]. However, not all families have the necessary the knowledge and some of them need help. Mattern et al. [7] found in a German study that organising a midwife for postpartum care might be very stressful for women, especially if many phone calls are necessary to achieve success. Sev- eral studies have shown that psychosocially and econom- ically disadvantaged families have limited access to postpartum care and use the provided services less fre- quently [8, 9]. Language barriers complicate the possibil- ities of organising care considerably [10, 11]. These families might need support for finding access to care. Midwifery networks that organise and guarantee out- patient postpartum care facilitate access and increase the use of the services [12]. If outpatient postpartum care consists of home visits after hospital discharge, this provides a great opportunity to gain deep insight into the domestic situation [13–15]. The early support of disadvantaged families requires a well-functioning net- work involving various professionals such as employees of youth and social services, psychologists, physicians and others [16]. Midwives can assume an important role in such networks by identifying psychosocially dis- advantaged families soon after birth and giving them follow up options such as early child support [17]. If psychosocially disadvantaged families find access to out of hospital postpartum care, a previous study showed that care was more extensive, because these families often need more support for the transition into parent- hood [18]. Although more extended postpartum care generated additional costs, the overall health insurance expenditure during the first month after birth for mothers and their children remained stable after imple- menting a midwifery network, because the health care costs for the infants declined [12]. This means that costs for postpartum care are a good investment, be- cause they prevent health problems and additional costs. Working in networks might also facilitate work scheduling for health professionals and enable a better use of their resources because the workload of postpar- tum care is irregular due to phases with high and low birth rates [19]. Hitherto, no study has investigated if midwifery net- works reach families who would struggle to find access to out of hospital postpartum care without this support. Furthermore, no previous study has researched women’s and midwives’ views of these services. Study design and participants We conducted a mixed method study design combining quantitative secondary analysis of routine data of Swiss independent midwives with telephone interviews with Page 3 of 12 Page 3 of 12 Grylka-Baeschlin et al. BMC Health Services Research (2020) 20:565 women who used the services of the midwifery network “Familystart Zurich” and focus group discussion with members of the network. Data of postpartum women living in the Canton of Zurich and giving birth in 2016 (n = 13,241) who either used the services of the network (n = 1544) to organise out of hospital postpartum care or organised it themselves (n = 11,697), were included in the secondary analysis. Women who used the services from the midwifery network with sufficient oral German language knowledge were eligible for the telephone in- terviews. Out of a total of 3108 users in 2016, 15 women were recruited evenly over a period of a year applying random selection stratified by parity, mode of birth, antenatal and postnatal registration as well as Swiss and foreign nationality. Midwives and nurses working in the network (n = 130) were invited to participate in the focus group discussion during the annual conference or were approached directly. Three midwives and two nurses consented to participate in the focus group discussion. quality insurance. To achieve full census, anonymised data is collected after oral information and consent (72, 017 women in 2016). Data is analysed and results are published annually [22]. The variables “residential Can- ton of the mother” and “infant’s birth date” allowed the identification of women living in the canton of Zurich and giving birth in 2016 (n = 13,241). In order to identify the users of the network “Familystart Zurich”, 147 mid- wives were asked to provide the ID-number of Familys- tart Zurich users who they cared for and who gave birth in 2016 in the routine data set of independent midwives. A total of 110 midwives responded to this request (74.8%), allowing the identification of 1791 women. This number was lower than the registered 3108 users of the network in 2016, but not all of them gave birth in 2016 (registration at the end of 2016 and infant birth date in the beginning of 2017) and data of women who were cared for by nurses were not included in the routine data of independent midwives. Data processing and analysis y From the routine data of independent midwives from the year 2016, women living in the canton of Zurich were extracted. Cases identified by members of the network as being “users of the network” were coded 1 and all the other cases were coded 0 for “women organising postpartum care themselves”. Users of the network were encouraged by antenatal care providers and hospitals to register for these services. Women organising care themselves searched the Internet for personal details of midwives and contacted them themselves. Risk factors for a vulnerable transition into parenthood with associated risks for impaired child development and for child abuse such as teen- age mother, single mother, no vocational education, jobless, poverty, migration, drug abuse, domestic vio- lence, multiples, preterm birth or child with congeni- tal malformation were investigated as single and cumulative factors [27, 28]. Descriptive statistics were computed according to the type of variables and study groups were compared using chi squared tests for categorical variables and Mann-Whitney-U-test for metric variables. Logistic regression modelling was carried out with the outcome variable “user of the network” yes or no. Potential predictors were vari- ables which were significantly associated with the out- come variable, namely the variables “age”, “parity”, “nationality”, “living in partnership”, “professional edu- cation”, “employment” and “mode of birth”. Backward elimination was done for variables which did not Development of the interview guides The interview guides were developed based on literature researches (supplementary files S1, S2). Studies investigat- ing patient satisfaction with the organisation of service providers revealed themes which were addressed during the telephone interviews with the users of the services of the network: access to the offers, waiting time, friendliness of contact persons and fees [24, 25]. The interview guide for the semi-structured focus group discussion was adapted from the telephone interviews with the women in order to collect information about similar topics from dif- ferent angles to enable triangulation. Additionally, job and occupational satisfaction of the midwives and nurses was addressed including the four themes of the subscales of the midwifery specific instrument from Turnbull et al. [26] professional satisfaction, professional support, client interaction and professional development. These themes were used as subjects of discussion. Ethics Qualitative data was collected by telephone and focus group interviews. The interviews were audio recorded and transcribed verbatim. The Swiss Federation of Midwives gave written permis- sion to use anonymised routine date for the purpose of the current study. Interview participants received oral and written study information and provided voluntarily written consent with the right to withdraw at any time. The Ethics Committee of the Canton of Zurich in Switzerland approved both, the quantitative (secondary analysis of routine data) and the qualitative part (tele- phone interviews, focus group discussion) of our study (BASEC-Nr. Req-2017-00192). Study design and participants Out of n = 1791 women, n = 1544 lived in the Canton of Zurich. Data collection Since 2005, routine data of independent midwives has been collected by the Swiss Federation of Midwives for Grylka-Baeschlin et al. BMC Health Services Research (2020) 20:565 Page 4 of 12 Page 4 of 12 persons, one midwifery researcher with German mother tongue and one language instructor with English mother tongue. remain significantly associated with the outcome vari- able in the model and age was kept in the model in- dependently of the significance of the association. The logistic regression model was computed with and without the variable “parity”, because parity showed 28.8% of missing values due to technical problems of the data collection tool [22]. Due to the importance of the variable and the statistically significant associ- ation with the outcome variable, sensitivity analysis with and without the variable “parity” were conducted and results including “parity” are presented. Quantita- tive analyses were done using Stata 13 (StataCorp, Tx, USA). Results Out of 3108 women referred in 2016 by the midwifery network Familystart Zurich to out of hospital postpar- tum care, 1544 were identified in the routine data of Swiss independent midwives as living in the canton of Zurich. Users of the network were compared with 11,697 women organising their postpartum care them- selves and living in the same canton. Swiss German interviews were transcribed in German as is common practice, because Swiss German is not a written language. Typical Swiss German words with no clear equivalent in German were maintained. The tran- scribed texts were analysed using content analysis fol- lowing the method of Mayring 2015 [29] and using the software Atlas.ti, version 8. Citations were linguistically polished in order to enhance comprehensibility and readability and translated into English if used for this publication. Translations were checked by two bilingual b Before giving birth, out of women, who were employed, n = 8556 c 28 8% i i l Sociodemographic and perinatal characteristics BMC Health Services Research (2020) 20:565 from Southern Europe (9.3% vs 5.0%, p < 0.001), Eastern Europe (17.4% vs 9.5%, p < 0.001), Africa (6.9% vs 2.0%, p < 0.001), Latin America (3.6% vs 1.9%, p < 0.001) and Asia (15.9% vs 6.3%, p < 0.001, Fig. 1). There was no sig- nificant difference in women coming from North Amer- ica (1.0% vs 0.8%, p = 0.341) and Oceania (0.3% vs 0.2%, p = 0.502) between study groups. Users of the services of the network had no vocational education significantly more often (25.7% vs 9.3%, p < 0.001) and were less often employed before giving birth (62.4% vs 75.5%, p < 0.001). However, if employed, they worked full time significantly more often (48.3% vs 36.8%, p < 0.001). from Southern Europe (9.3% vs 5.0%, p < 0.001), Eastern Europe (17.4% vs 9.5%, p < 0.001), Africa (6.9% vs 2.0%, p < 0.001), Latin America (3.6% vs 1.9%, p < 0.001) and Asia (15.9% vs 6.3%, p < 0.001, Fig. 1). There was no sig- nificant difference in women coming from North Amer- ica (1.0% vs 0.8%, p = 0.341) and Oceania (0.3% vs 0.2%, p = 0.502) between study groups. Users of the services of the network had no vocational education significantly more often (25.7% vs 9.3%, p < 0.001) and were less often employed before giving birth (62.4% vs 75.5%, p < 0.001). However, if employed, they worked full time significantly more often (48.3% vs 36.8%, p < 0.001). and child with congenital malformation (1.3% vs 0.8%, p = 0.045, Table 2). There was no significant difference regarding young mothers < 18 years, multiparous women < 20 years, drug abuse, domestic violence and suspected child abuse as well as depression. Users of the network also had no risk factor less often (57.4% vs 77.8%, p < 0.001) but more often one (29.2% vs 16.5%, p < 0.001), two (9.3% vs 4.3%, p < 0.001) and three or more risk factors (4.2% vs 1.5%, p < 0.001) than women organising postpartum care themselves. Risk factors Compared to women organizing out of hospital postpar- tum care themselves, users of the midwifery network had the following social and medical risk factors for vul- nerable transition into parenthood significantly more often: single mother (3.0% vs 1.5%, p < 0.001), no voca- tional education (25.7% vs 9.3%, p < 0.001), jobless (5.6% vs 3.0%, p < 0.001), poverty (1.7% vs 0.3%, p < 0.001), mi- gration (7.1% vs 3.3%, p < 0.001), multiples (2.9% vs 1.8%, p = 0.007), preterm birth (7.7% vs 5.3%, p < 0.001) Predictors for the use of the services of the midwifery network Additionally, women using the services of the midwif- ery network were significantly more often first-time mothers (57.0% vs 49.3%, p < 0.001), had their second child less often (29.2% vs 37.9%, p < 0.001) but similarly often their third child or more (13.8% vs 12.8%, p = 0.342) compared to women organizing their postpartum care themselves. Furthermore, users of the network gave birth spontaneously significantly less often (48.6% vs 56.2%, p < 0.001), had an instrumental vaginal birth similarly often (9.05% vs 9.6%, p = 0.408) but a caesarean section more often (42.4% vs 34.2%, p < 0.001). Multivariable analyses with the outcome variable “user of the network” yes or no were computed with and without the variable “parity” because of missing values for this par- ameter. Results of the logistic regression including the variable parity are shown in Table 3. Women with foreign nationality (OR 2.36, 95% CI [2.04–2.73], p < 0.001), with- out vocational education (OR 1.89, 95% CI [1.56–2.29], p < 0.001), who were not employed (OR 1.28, 95% CI [1.09–1.51], p = 0.002) or had given birth by caesarean sec- tion (OR 1.38, 95% CI [1.20–1.59], p < 0.001) were more likely to use the services of the midwifery network to gain access to out of hospital postpartum care. More highly ed- ucated women (OR 0.85, 95% CI [0.72–0.99], p = 0.003), having the second child (OR 0.62, 95% CI [0.53–0.73], p < 0.001) or their third child and more (OR 0.79, 95% CI [0.64–0.98], p = 0.035) were less likely to be users of the midwifery network. Sociodemographic and perinatal characteristics The comparison with women who organised midwifery postpartum care themselves showed that users of the network were slightly younger (median age: 32 years vs 33 years, p < 0.001) and less often of Swiss nationality (31.6% vs 58.7%, p < 0.001, Table 1). Regarding foreign nationalities grouped by geographic regions and conti- nents, users of the midwifery network came slightly but not significantly less often from North-western Europe (14.2% vs 15.7%, p = 0.135), but significantly more often Table 1 Sociodemographic characteristics of users of the midwifery network compared to women, who organised postpartum care themselves Characteristics Whole study population n = 13,241 Users of the midwifery networka n = 1544 Women organising postpartum care themselvesa n = 11,697 p- value Sociodemographic characteristics Age in years, md (min-max) 33 (16–59) 32 (17–56) 33 (16–59) < 0.001 Swiss nationality, n (%) 7202 (55.6) 479 (31.6) 6723 (58.7) < 0.001 Living in partnership, (n (%) 12,682 (98.4) 1445 (97.0) 11,237 (98.5) < 0.001 Highest vocational education < 0.001 No voc. Education, n (%) 1408 (11.2) 374 (25.7) 1034 (9.3) Apprenticeship/high school education, n (%) 5793 (46.2) 587 (40.3 5206 (47.0) Higher education/university, n (%) 5333 (42.6) 496 (34.0) 4837 (43.7) Employed, n (%) 9350 (74.0) 927 (62.4) 8423 (75.5) < 0.001 Full time employedb, n (%) 3243 (37.9) 406 (48.3) 2837 (36.8) < 0.001 Obstetric and perinatal characteristic Parityc < 0.001 First child, n (%) 4741 (50.3) 682 (57.0) 4059 (49.3) Second child, n (%) 3466 (36.8) 350 (29.2) 3116 (37.9) Third child and more, n (%) 1218 (12.9) 165 (13.8) 1053 (12.8) Mode of birth < 0.001 Spontanous vaginal, n (%) 6984 (55.2) 748 (48.6) 6236 (56.2) Instrumental vaginal, n (%) 1208 (9.6) 138 (9.0) 1070 (9.6) Caesarean section, n (%) 4452 (35.2) 652 (42.4) 3800 (34.2) a All living in the canton of Zurich b Before giving birth out of women who were employed n = 8556 Table 1 Sociodemographic characteristics of users of the midwifery network compared to women, who org themselves hic characteristics of users of the midwifery network compared to women, who organised postpartum care Page 5 of 12 Page 5 of 12 Grylka-Baeschlin et al. Organising midwifery care them spoke German without any accent and were from Switzerland, Germany or Austria. One third of the inter- viewed users of the midwifery network spoke broken to fluent German with an accent and were from Kenya, Angola, Slovakia, Belarus and Poland. Despite addressing equal numbers of women registering before and after having given birth to ask if they would like to be inter- viewed, 86.7% (n = 13) of the participants registered dur- ing pregnancy. Nearly half of the women mentioned stressful situations: Migration, multiples miscarriages and preterm birth in history or age over 50 years. Add- itionally, the majority of women (n = 12) noted peculiar- ities or pathologies during pregnancy, birth or the postpartum period such as bleeding during pregnancy, premature labour, gestational diabetes, pelvic floor prob- lems, twins, foetal tachycardia, unplanned repeat caesar- ean section, emergency caesarean section, vaginal birth after caesarean section, traumatic birth, referral to neo- natal intensive unit or increased weight loss of the new- born. Not all interviewed women had information about the possibilities of out of hospital postpartum care and how to organise it. “(…) I waited quite a long time, because I did not really know how to find a midwife (…)” “(…) I waited quite a long time, because I did not really know how to find a midwife (…)” “(…) I waited quite a long time, because I did not really know how to find a midwife (…)” The search for a midwife proceeded very differently. Some users of the midwifery network were registered by the hospital without understanding how the registration and the organisation of midwifery care took place. Women registering themselves for the services of the midwifery network reported very different experiences trying to organise out of hospital postpartum care be- forehand. Some of them were very frustrated while try- ing to organise it themselves or experienced difficulties organising postpartum care for a previous child. “… I looked on the special websites with addresses of midwives and (…) it turned out to be very difficult; so sometimes (…) I did not even receive a response and sometimes “I’m sorry, I’m fully booked, I have no capacity” (…)” “(…) I asked ten midwives, if they would have time and all of them declined (…)”. Women’s satisfaction and benefits Participants of the 15 telephone interviews were on aver- age 35.9 years (range 27–57 years) old. Two-thirds of Fig. 1 Nationalities grouped by geographic regions in users of the midwifery network compared to women, who organised postpartum care themselves Fig. 1 Nationalities grouped by geographic regions in users of the midwifery network compared to women, who organised postpartum care themselves Grylka-Baeschlin et al. BMC Health Services Research (2020) 20:565 Page 6 of 12 Table 2 Risk factors in users of the midwifery network compared to women, who organised postpartum care themselves Risk factor/number of risk factors Whole study population n = 13,241 Users of the midwifery networka n = 1544 Women organising postpartum care themselvesa n = 11,697 p-value Risk factor Young mother < 18 years, n (%) 37 (0.3) 4 (0.3) 33 (0.3) 0.872 Multiparous women < 20 years, n (%) 5 (0.1) 0 5 (0.1) 1.000b Single mother, n (%) 213 (1.7) 44 (3.0) 169 (1.5) < 0.001 No vocational education, n (%) 1408 (11.2) 374 (25.7) 1′034 (9.3) < 0.001 Jobless, n (%) 375 (3.4) 76 (5.6) 299 (3.0) < 0.001 Poverty, n (%) 46 (0.5) 18 (1.7) 28 (0.3) < 0.001 Migration, n (%) 353 (3.8) 78 (7.1) 275 (3.3) < 0.001 Drug abuse, n (%) 3 (0.0) 1 (0.1) 2 (0.0) 0.310b Domestic violence or suspected child abuse, n (%) 20 (0.2) 3 (0.3) 17 (0.2) 0.501b Depression, n (%) 149 (1.6) 23 (2.1) 126 (1.5) 0.147 Multiples, n (%) 259 (2.0) 44 (2.9) 215 (1.8) 0.007 Preterm birth, n (%) 735 (5.6) 119 (7.7) 616 (5.3) < 0.001 Child with congenital malformation, n (%) 113 (0.9) 20 (1.3) 93 (0.8) 0.045 Number of risk factors No risk factor, n (%) 4692 (75.2) 456 (57.4) 4236 (77.8) < 0.001 One risk factor, n (%) 1129 (18.1) 232 (29.2) 897 (16.5) < 0.001 Two risk factors, n (%) 307 (4.9) 74 (9.3) 233 (4.3) < 0.001 Three or more risk factors, n (%) 112 (1.8) 33 (4.2) 79 (1.5) < 0.001 a All living in the canton of Zurich b Fisher’s exact test Organising midwifery care Mode of birth Instrumental vaginal birth Caesarean section (Reference category spontaneous vaginal birth) Service-users who tried to organise out of hospital postpartum care themselves highly appreciated the services and were very grateful about the easy and straightforward support which saved time, worries and stress. It was an issue for some women that they could not choose the midwife themselves or influence the alloca- tion when using the services of the midwifery network. Thus, the midwife remained anonymous until postpar- tum care started. “(…) I can really express my thanks again for the straightforward and quick help and also that I had such a lovely midwife (…). It is really amazing that this offer exists (…). It was a great help then and decreased my worries and saved me time and for me, it was really great. I could actually not believe that it was so straightforward and easy and free. And my husband thought (…) yes, why did you call so many (midwives) yourself.” “(…) if I had had the possibility to choose, I would have looked for a midwife who I could at least have seen a picture of or perhaps have spoken to on the telephone”. However, other women did not have concrete ideas or expectations about the midwife and were not challenged with the impersonal organisation of care. “I did not look for (…) a particular person because I did not have any previous experience (…). But it was important for me to have a midwife with profes- sional experience and this was actually my only criterion.” The contact with the midwifery network mostly took place by e-mail. Women who had contact with the office of the network reported that the communication was very friendly and professional. “(…) I think it was very professional and kind and that/that was enough for me, so, it was just good like this.” The services of the midwifery network Some participants of the telephone interviews were reg- istered with the midwifery network by the hospital staff. These women did not have any difficulty accessing the services and some of them did not realise how the or- ganisation of the out of hospital postpartum care proceeded. Organising midwifery care The statements of the women on their satisfaction with the services of the network and on the benefits of its use were grouped into three themes: “Organising midwifery care”, “The services of the midwifery network” and “Postpartum care”. Grylka-Baeschlin et al. BMC Health Services Research (2020) 20:565 Page 7 of 12 Grylka-Baeschlin et al. BMC Health Services Research (2020) 20:565 Page 7 of 12 Table 3 Logistic regression: Predictors for the use of the services of the midwifery network, n = 8384 Predictor Odds Ratio 95% confidence interval p-value Age in years 0.99 0.98–1.00 0.143 Nationality Foreign nationality (Reference category Swiss) 2.36 2.04–2.73 < 0.001 Highest vocational education No vocational education 1.89 1.56–2.29 < 0.001 Higher education/university (Reference category apprenticeship/ high school education) 0.85 0.72–0.99 0.033 Employed Not employed 1.28 1.09–1.51 0.002 Parity Second child 0.62 0.53–0.73 < 0.001 Third child or more (Reference category first child) 0.79 0.64–0.98 0.035 Mode of birth Instrumental vaginal birth 0.97 0.77–1.24 0.832 Caesarean section (Reference category spontaneous vaginal birth) 1.38 1.20–1.59 < 0.001 Postpartum care Satisfaction with postpartum care was very high for the participants of the telephone interviews and for many women, the midwife evolved into an important reference person. “It was good, even perfect (…) she organised me (…) the midwife, and it was ok (…). I did not want to stay in hospital long because I had a caesarean section.” “(…) she (the midwife) was very (…) empathic and friendly. And she was kind of a lifesaver for me. She Page 8 of 12 Grylka-Baeschlin et al. BMC Health Services Research (2020) 20:565 was (uh) (..) I have to cry when I think about her, because it was (uh) a really difficult start for me”. “I see a clear advantage that I get an overview of women (who are looking for a midwife) and that it states where they live, their due date and some additional information. This already helps me to (…) prepare my schedule.” Women with migration background recognised the midwife as a cultural mediator. “(…) and for me, it is good, because I profited from two experiences, from my mother’s and from the midwife’s (…) and I am very happy about this.” “(…) and for me, it is good, because I profited from two experiences, from my mother’s and from the midwife’s (…) and I am very happy about this.” However, the introduction of new technologies re- quired some effort for the members. “This is a technical problem; how does this app really work. Why did they have to change this (…).” Some women also appreciated it if the midwife respected their individual boundaries. However, women needed confirmation that they handled the new situation well. The midwifery network is also active in the interface management with the hospitals and the follow-up support offered to families. Members appreciated the contacts of the network with the hospitals and the follow-up offers but would appreciate if the network would operate as an ombudsman service for feedbacks to the hospital about women and their children who, according to midwives’ assessment, left the hospital in a poor state of health. “I have very positive memories. I was very happy that she (the midwife) was not intrusive (…) because, yes, I was a little bit afraid about people who know better. But she was (…) very pleasant. She saw that we were both good together (…). Use of resources Th b i i The beginning of care for women who register before giving birth may vary by several weeks around the due date because of the uncertainty about the birth date of the baby. This often leads to an imbalanced workload. Participants of the interview emphasised that women who register to the network at short notice enabled gaps in their workload to be filled. Midwives’ and nurses’ satisfaction and benefits Three midwives and two nurses specialising in postpar- tum care participated in the focus group discussion. Their mean age was 52.4 years (43–57 years) and they had 23.3 years of professional experience (16–30 years). Participants had been self-employed for an average of 12 years (1–26 years) and in 2016, cared for 132 women (10–250 women) on average. “(…) you can fill all your work gaps very well. So, you have almost no risks anymore. You even need to check that you do not work too much.” Being able to have an overview of women who are looking for a midwife provides the possibility to choose those close to the midwife’s home or to other patients and therefore to keep routes short. The statements of the midwives and nurses were grouped into four themes: “The membership in the net- work”, “Use of resources”, “Caring for users of the network” and “Job satisfaction”. “I have almost no travel time anymore (to visit women). I walk a lot (…). Thus, you can save a lot of time.” The membership in the network The network operates a mobile application providing an overview of women who are looking for a midwife. Members of the network appreciated that this facilitated their work organisation. Postpartum care And she said that everything worked out very well (…) and yes (…) it was very pleasant.” “We created a new transfer form (for the follow-up offers) and we work very well with this.” “(…) if it were possible to put the complaints (to the hospitals) to a neutral ombudsman service, without annoying people or having to go to different hospitals myself.” “We created a new transfer form (for the follow-up offers) and we work very well with this.” An important point was the accessibility and reliability of the midwife. Women became nervous if it was diffi- cult to reach the midwife or if she did not adhere to meeting times and was late. “(…) if it were possible to put the complaints (to the hospitals) to a neutral ombudsman service, without annoying people or having to go to different hospitals myself.” “… this is the only thing I have to say which was not so good. Because, after the baby’s birth, my husband sent a SMS and called the number which we received. And for two-three days, we did not get an answer.” “… this is the only thing I have to say which was not so good. Because, after the baby’s birth, my husband sent a SMS and called the number which we received. And for two-three days, we did not get an answer.” Caring for users of the network Members of the midwifery network observed that women using the services of the network differed from Page 9 of 12 Page 9 of 12 Grylka-Baeschlin et al. BMC Health Services Research (2020) 20:565 women organising postpartum care themselves. Users of the network often did not have knowledge about the Swiss maternity care system. families who might have been challenged to organise postpartum care themselves in a health system, in which it is normal practice to do so. Additionally, women ap- preciated the services very much and confirmed the need of support for organising postpartum care. Being a member of the network enabled midwives and nurses to better organise their work and use their resources and job satisfaction of the professionals was high. “Hence, there are certainly more vulnerable families (…) because the others, who can find a midwife themselves, they realised that it would be good to have follow-up-care.” “But even certain women, (…) who have a higher educational level (…) do not know it (that they should organise care themselves).” “Hence, there are certainly more vulnerable families (…) because the others, who can find a midwife themselves, they realised that it would be good to have follow-up-care.” “But even certain women, (…) who have a higher educational level (…) do not know it (that they should organise care themselves).” Users of the network differed from women organis- ing out of hospital postpartum care themselves and were more frequently primiparae, of foreign national- ity, had vocational training less often, were more fre- quently unemployed and gave birth more often by caesarean section. They also showed one or several risk factors for a vulnerable transition into parenthood leading to an unfavourable environment for child de- velopment more often. Our results could also be com- pared with unpublished cumulative data from 2013 to 2015 of the general population provided from the stat- istical office of the canton of Zurich. In the population of women of childbearing age, only 39.8% were of for- eign nationality compared with 68.4% of the users of the network. The proportion of foreigners in the users of the network was even higher than the one in the largest hospital (University Hospital Zurich, personal communication, 2017) which had a contract with the network (68.4% vs 59.4%). Job satisfaction The midwives and nurses were generally very satisfied with their work as self-employed midwives but had diffi- culties to distinguish between general job satisfaction and satisfaction due to their participation in the net- work. Services of the network such as enabling the pos- sibility to choose women who lived near to each other or close to the midwife’s home and therefore, avoiding long journeys for the home visits seem to increase satisfaction. “… I’m very, very satisfied, I have to say (…). It is also very nice to travel by bike…. It is great to ride the streets with the e-bike…”. “… I’m very, very satisfied, I have to say (…). It is also very nice to travel by bike…. It is great to ride the streets with the e-bike…”. Caring for users of the network The difference between users of the network and other women (68.4% vs 44.4%) was also higher than the general difference be- tween city and canton of Zurich (39.8% versus 33.5%) (Statistical Office Canton of Zurich, Cumulative data 2013-2015, unpulished data). This was especially im- portant because more than half of the users of the network lived in the city. Additionally, the high pro- portion of women originating from Southern and East- ern Europe as well as from Asia and Africa showed that users of the network often had cultural back- grounds which were fundamentally different from the Central European one in which they lived. Comparing the highest vocational education of the users of the network with the general population of women of childbearing age showed a similar picture. The propor- tion of woman without vocational education was 13.7% in the city of Zurich and 16.4% in the whole canton of Zurich (inclusively city) (Statistical Office Canton of Zurich, Cumulative data 2013-2015, unpu- lished data) compared to 25.7% in the users of the network. The proportion of caesarean section of users of the network (43.2%) was also higher than the aver- age in the three contract hospitals (39.0%) (Familystart Zurich, caesarean section rate in contract hospitals 2016, personal communication 2017) and higher than in most city areas of Zurich (32.6–44.5%) and higher than the cantonal average (37 2%) [30] Midwives and nurses experience a sincere gratitude from users of the network but also a great responsibility when caring for vulnerable families. Home visits allow a deep insight into their family lives and enable for ex- ample observation of older siblings’ behaviour. “They [the women] are very grateful if you say “yes (…) I will come tomorrow, no problem and I will bring a breast pump” and then you know that they already feel a bit better (…). And because they are desperately looking for a midwife and sometimes when they leave the hospital, they do not know if somebody will come the next day (…) they are really grateful.” “We have an insight into these families and see how parents care for the siblings. We can observe for ex- ample if a three-year-old (sibling of the baby) is sit- ting in front of the television all day.” Midwives were also confronted with language difficul- ties and would have appreciated the support of inter- preters with cultural mediation skills. Discussion Our study showed the great value of organised and guar- anteed postpartum care for socially disadvantaged fam- ilies and midwives. The network was able to reach Page 10 of 12 Page 10 of 12 Grylka-Baeschlin et al. BMC Health Services Research (2020) 20:565 Previous studies showed that psychosocial disadvan- tages and language barriers prevent women from having access to postpartum care [8–11]. Our study findings in- dicate that burdened families, who were more often of foreign nationality, with no vocational training and had more risk factors for impaired transition into parent- hood, might have more difficulty finding access to care but offering them support, enables it. This is especially important in the vulnerable period after giving birth to promote a safe transition into parenthood [2–4]. Erdin et al. [18] found in a secondary analysis of routine data of Swiss independent midwives of the whole country that women with risk factors needed even more care and home visits. Our analyses showed that despite this add- itional need for support, burdened families who often do not have enough knowledge about the health care sys- tem, would have fallen through the cracks of maternity care if they could not have profited from the services of the network. This confirms the findings of Wilcox et al. [10] that socially and economically vulnerable families did not use the proposed postpartum care services. Moreover, our findings were also in line with the ones of a German study which found that women with psycho- social problems and limited literacy are overwhelmed by organising midwifery care themselves [8]. It seems there- fore very important that access to midwifery networks which support families in organising care should be fa- cilitated and be promoted by antenatal care providers and hospitals. differences between study groups was robust despite this small bias. Missing data is a known problem in observa- tional studies [31]. Due to technical problems during data collection, the proportion was especially high for the variable parity. The logistic regression could there- fore not be performed with a complete dataset. However, we could not identify any pattern in missing data which was related to the distinction between study groups, which reduces the risk of biased results. Sample sizes for the telephone interviews with users of the network and for the focus group discussion were small and results might not be fully representable for the study popula- tion. Discussion The stratified selection of potential candidates for the telephone interviews aimed for a heterogeneous study sample. Nevertheless, women registering during pregnancy and those speaking German well participated more often. Interviewed women were therefore not all socially disadvantaged but nevertheless, provided valu- able information about the benefits of the services of the midwifery network. A further limitation was that mid- wives and nurses participating in the focus group discus- sion might have been the most engaged ones with positive views. Furthermore, discussing job satisfaction in a focus group might have prevented some participants from communicating all their concerns. Additionally, the health professionals cared for varying numbers of women per year leading to very different working situa- tions. The workload might have influenced their state- ments because a higher one might have been associated with increased stress but maybe also with increased work engagement. Many midwives in Switzerland work very independ- ently and are not organised in networks [19]. They often bear the risk of imbalanced workloads and burdens of difficult care situations themselves. The statement of midwives and nurses in the current study showed that being a member of a network helps to schedule work. However, the participants of the focus group discussion mentioned possibilities for improvement regarding the support of the network, such as their wish to have an ombudsman service for difficult feedbacks to the hospi- tals. Further research is needed to investigate which kind of support is necessary to ease the burden of postpartum health care providers, especially if they care for psycho- socially disadvantaged families. Conclusion Our study showed that through the services of a midwif- ery network psychosocially disadvantaged families who need support for a good transition into parenthood could be reached. They might not have been able to or- ganise postpartum care themselves and might have fallen through the safety net of the supply network without this support. Guaranteed postpartum care eases the bur- den on families and reduces stress. As a consequence, families appreciated the services of the midwifery net- work very much. The network also facilitated midwives and nurses to schedule their work and enabled a better use of their resources. Hence, our study showed on the one hand, that the responsibility to organise care should not be left to the families, because especially psycho- socially challenged ones are overburdened with this task. Access to such networks should be facilitated and pro- moted by antenatal care providers and hospitals. On the other hand, the study also demonstrated that working together in networks has many advantages for the mid- wives. Midwifery networks have therefore benefits for families as well as for health professionals. Future studies Strengths of our study were the relatively large data set for the comparison of women using the services of the midwifery network with those organising postpartum care themselves. Nevertheless, it was not possible to identify all users in the routine data of Swiss independ- ent midwives and for some variables, the proportion of missing data was high. Users of the network who were not identified but gave birth in 2016 were wrongly allo- cated to the study group including women who orga- nised care themselves. This bias would only have weakened our results which indicates that the significant Page 11 of 12 Page 11 of 12 Grylka-Baeschlin et al. BMC Health Services Research (2020) 20:565 should investigate midwives’ needs for support when caring for burdened families such as access to interpreter services. should investigate midwives’ needs for support when caring for burdened families such as access to interpreter services. Received: 1 October 2019 Accepted: 24 May 2020 Availability of data and materials Th d d d/ l The datasets generated and/or analysed during the current study are not publicly available due to confidentiality of data but are available from the corresponding author on reasonable request. 14. Meier Magistretti C, Villiger S, Luyben A, Varga I. Qualität und Lücken der nachgeburtlichen Betreuung. Eine Studie zu postnatal care im Rahmen der COST-Aktion IS907: ‘Childbirth: cultures, concerns and consequences’ (Quality and gaps in postpartum care. A study about postpartum care within the COST-Action IS907: ‘Childbirth: cultures, concerns and consequences’). Hochschule Luzern Soziale Arbeit; 2014. https://www.hslu. ch/de-ch/hochschule-luzern/forschung/projekte/detail/?pid=874. Accessed 30 Sept 2019. Acknowledgments 6. Olson T, Bowen A, Smith-Fehr J, Ghosh S. Going home with baby: innovative and comprehensive support for new mothers. Prim Health Care Res Dev. 2018;27:1–6. 6. Olson T, Bowen A, Smith-Fehr J, Ghosh S. Going home with baby: innovative and comprehensive support for new mothers. Prim Health Care Res Dev. 2018;27:1–6. g Many thanks to Lottery Fund of the Canton of Zurich for funding this study and to the management of Familystart Zurich for launching and supporting it. We also thank the Swiss independent midwifes for collecting routine data and the Swiss Federation of Midwives for agreeing to use this routine data for our study. Additionally, we sincerely thank the participants of the telephone interviews and focus group discussion for their time and valuable information. Many thanks to Lottery Fund of the Canton of Zurich for funding this study and to the management of Familystart Zurich for launching and supporting it. We also thank the Swiss independent midwifes for collecting routine data and the Swiss Federation of Midwives for agreeing to use this routine data for our study. Additionally, we sincerely thank the participants of the 7. Mattern E, Lohmann S, Ayerle GM. Experiences and wishes of women regarding systemic aspects of midwifery care in Germany: a qualitative study with focus groups. BMC Pregnancy Childbirth. 2017;17(1):389. 7. Mattern E, Lohmann S, Ayerle GM. Experiences and wishes of women regarding systemic aspects of midwifery care in Germany: a qualitative study with focus groups. BMC Pregnancy Childbirth. 2017;17(1):389. telephone interviews and focus group discussion for their time and valuable information. 8. DiBari JN, Yu SM, Chao SM, Lu MC. Use of postpartum care: predictors and barriers. J Pregnancy. 2014;2014:530769. 8. DiBari JN, Yu SM, Chao SM, Lu MC. Use of postpartum care: predictors and barriers. J Pregnancy. 2014;2014:530769. g y 9. Wilcox A, Levi EE, Garrett JM. Predictors of non-attendance to the postpartum follow-up visit. Matern Child Health J. 2016;20(Suppl 1):22–7. Funding h d Funding The study was funded by the Lottery Fund of the Canton of Zurich. The study was funded by the Lottery Fund of the Canton of Zurich. Ethics approval and consent to participate The Swiss Federation of Midwives gave written permission to use anonymised routine date for the purpose of the current study. Interview participants received oral and written study information and provided voluntarily written consent with the right to withdraw at any time. The Ethics Committee of the Canton of Zurich in Switzerland approved both, the quantitative (secondary analysis of routine data) and the qualitative part (telephone interviews, focus group discussion) of our study (BASEC-Nr. Req- 2017-00192). 15. Perrenoud P. Construire des savoirs issus de l’expérience à l’ère de l’évidence- base médicine: Une enquête anthropologique auprès de sages-femmes indépendantes en suisse romand (construct knowledge about the experience in the age of evidence based medicine: an anthropological study with independent midwives in the French part of Switzerland. 2016. https://serval. unil.ch/resource/serval:BIB_A8B46DD35316.P001/REF. Accessed 30 Sept 2019. 15. Perrenoud P. Construire des savoirs issus de l’expérience à l’ère de l’évidence- base médicine: Une enquête anthropologique auprès de sages-femmes indépendantes en suisse romand (construct knowledge about the experience in the age of evidence based medicine: an anthropological study with independent midwives in the French part of Switzerland. 2016. https://serval. unil.ch/resource/serval:BIB_A8B46DD35316.P001/REF. Accessed 30 Sept 2019. Abbreviations CI: Confidence interval; DRG: Diagnosis related groups; OR: Odds ratio; vs: versus 5. Farhat R, Rajab M. Length of postnatal hospital stay in healthy newborns and re-hospitalization following early discharge. N Am J Med Sci. 2011;3(3): 146–51. 5. Farhat R, Rajab M. Length of postnatal hospital stay in healthy newborns and re-hospitalization following early discharge. N Am J Med Sci. 2011;3(3): 146–51. Consent for publication Not applicable. 16. Hafen M. Interdisziplinarität in der Frühen Förderung, Notwendigkeit, Herausforderung und Chance. Frühförderung interdisziplinär (Interdisciplinarity in early learning programmes, necessity, challenges and opportunity). 2013. https://www.reinhardt-journals.de/index.php/fi/article/ view/1753. Accessed 30 Sept 2019. 16. Hafen M. Interdisziplinarität in der Frühen Förderung, Notwendigkeit, Herausforderung und Chance. Frühförderung interdisziplinär (Interdisciplinarity in early learning programmes, necessity, challenges and opportunity). 2013. https://www.reinhardt-journals.de/index.php/fi/article/ view/1753. Accessed 30 Sept 2019. Authors’ contributions 10. Almeida LM, Caldas J, Ayres-de-Campos D, Salcedo-Barrientos D, Dias S. Maternal healthcare in migrants: a systematic review. Matern Child Health J. 2013;17(8):1346–54. All authors (SG-B, CI, RE, JP-M) were involved in the planning of the study. RE ex- tracted the data from routine data of independent midwives, collected by the Swiss Federation of Midwives. SG-B conducted the telephone interviews and the focus group discussions, analysed the data and drafted the manuscript. RE advised on the quantitative and JP-M on the qualitative analyses. All authors reviewed and revised the manuscript. The author(s) read and approved the final manuscript. 11. Origlia Ikhilor P, Hasenberg G, Kurth E, Asefaw F, Pehlke-Milde J, Cignacco E. Communication barriers in maternity care of allophone migrants: experiences of women, healthcare professionals, and intercultural interpreters. J Adv Nurs. 2019;7510:2200–10. 12. Zemp E, Signorell A, Kurth E, Reich O. Does coordinated postpartum care influence costs? Int J Integr Care. 2017;17(1):7. 12. Zemp E, Signorell A, Kurth E, Reich O. Does coordinated postpartum care influence costs? Int J Integr Care. 2017;17(1):7. 13. König C, Pehlke-Milde J. Bestandesaufnahme des Betreuungs-, Beratungs- und Unterstützungsangebots für Wöchnerinnen in der Schweiz: Schlussbericht. Bern: zuhanden des Bundesamts für Gesundheit (BAG) (Inventory of the care, counselling and support offers for women during the postpartum period in Switzerland: final report. Bern: for the attention of the Federal Office of Public Health (FOPH); 2010. https://www.aramis.admin.ch/ Default.aspx?DocumentID=45462&Load=true. Accessed 1 Apr 2020. Supplementary information Supplementary information Supplementary information accompanies this paper at https://doi.org/10. 1186/s12913-020-05359-3. Additional file 1. Additional file 2. Supplementary information Supplementary information accompanies this paper at https://doi.org/10. 1186/s12913-020-05359-3. Additional file 1. Additional file 2. Supplementary information Supplementary information accompanies this paper at https://doi.org/10. 1186/s12913-020-05359-3. Additional file 1. Additional file 2. pp y Supplementary information accompanie y Supplementary information accompanies this paper at https://doi.org/10. 1186/s12913-020-05359-3. 2. 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Safe start at home: what parents of newborns need after early discharge from hospital - a focus group study. BMC Health Serv Res. 2016;16:82. Competing interests The authors declare that they have n Competing interests The authors declare that they have no competing interests. The authors declare that they have no competing interests. 17. Ayerle GM, Makowsky K, Schucking BA. Key role in the prevention of child neglect and abuse in Germany: continuous care by qualified family midwives. Midwifery. 2012;28(4):e529. 17. Ayerle GM, Makowsky K, Schucking BA. Key role in the prevention of child neglect and abuse in Germany: continuous care by qualified family midwives. Midwifery. 2012;28(4):e529. Author details 1Research Unit for Midwifery Science, ZHAW Zurich University of Applied Sciences, Technikumstr. 81, CH-8401 Winterthur, Switzerland. 2Familystart Zurich, Thurgauerstrasse 39, CH-8050 Zurich, Switzerland. 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An ethnographic investigation of the maternity healthcare experience of immigrants in rural and urban Alberta, Canada. BMC Pregnancy Childbirth. 2016;16:20. 25. Polluste K, Kallikorm R, Meiesaar K, Lember M. Satisfaction with access to health services: the perspective of Estonian patients with rheumatoid arthritis. ScientificWorldJournal. 2012;2012:257569. 26. Turnbull D, Reid M, McGinley M, Sheilds NR. Changes in midwives’ attitudes to their professional role following the implementation of the midwifery development unit. Midwifery. 1995;11(3):110–9. 27. Sentenac M, Johnson S, Charkaluk M-L, Sëppanen A-V, Aden U, Cuttini M, et al. Maternal education and language development at 2 years corrected age in children born very preterm: results from a European population- based cohort study. J Epidemiol Community Health. 2020;74(4):346–53. 28. Puls HT, Anderst JD, Bettenhausen JL, Clark N, Krager M, Markham JL, et al. Newborn Risk Factors for Subsequent Physical Abuse Hospitalizations. 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https://openalex.org/W2029375921	http://uu.diva-portal.org/smash/get/diva2:607789/FULLTEXT01	English	null	On the Stator Slot Geometry of a Cable Wound Generator for Hydrokinetic Energy Conversion	The scientific world journal/TheScientificWorldjournal	2,015	cc-by	4,340	1. Introduction designs for wind power [7], wave power [8], and the high voltage Powerformer [9].h The permanent magnet geometry and stator slot geometry are important design parameters in the magnetic circuit of an electrical machine. In addition, utilizing cable winding presents new stator slot geometries that have not previously been studied in great detail. In this paper, the stator slot design of a cable wound permanent magnet synchronous generator for hydrokinetic energy conversion is studied using finite element (FE) simulations. The importance of changes on the millimetre scale in the stator slot geometry is discussed based on the practical experience from the design and the assembly of two prototypes. The starting point, or reference geometry, is that of the first prototype presented in [10], rated at 5 kW and 150 V at 10 rpm. As with most early prototypes, the generator design at hand can most likely be improved in many aspects. In this study, however, the focus is on possible performance gains by changing the stator slot geometry in the existing design. In order to incorporate practical experience from construction of two prototypes, the influence of one parameter at a time is discussed rather than a traditional optimization study. Such improvements would likely be relevant even if other parameters in the design would be changed. The generator is designed to be directly connected to a fixed pitch vertical axis turbine, operating at both variable speed and power to electrically control the tip speed ratio of the turbine [1]. The generator is also intended to efficiently brake the turbine at water velocities above nominal operation, implying that a low load angle at nominal operation is preferable. Other direct drive designs for tidal turbines have been proposed recently; see, for instance, [2–6], but as the focus is on the stator slot geometry, the work presented here is more closely related to studies on other cable wound machine The Swedish Centre for Renewable Electric Energy Conversion, Division of Electricity, The ˚Angstr¨om Laboratory, Uppsala University, P.O. Box 534, 751 21 Uppsala, Sweden The Swedish Centre for Renewable Electric Energy Conversion, Division of Electricity, The ˚Angstr¨om Laboratory, Uppsala University, P.O. Box 534, 751 21 Uppsala, Sweden Correspondence should be addressed to Sandra Eriksson; sandra.eriksson@angstrom.uu.se Received 17 June 2014; Accepted 5 October 2014 Academic Editor: Linni Jian Copyright © 2015 M˚arten Grabbe et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The stator slot geometry of a cable wound permanent magnet synchronous generator for hydrokinetic energy conversion is evaluated. Practical experience from winding two cable wound generators is used to propose optimized dimensions of different parts in the stator slot geometry. A thorough investigation is performed through simulations of how small geometrical changes alter the generator performance. The finite element method (FEM) is used to model the generator and the simulations show that small changes in the geometry can have large effect on the performance of the generator. Furthermore, it is concluded that the load angle is especially sensitive to small geometrical changes. A new generator design is proposed which shows improved efficiency, reduced weight, and a possibility to decrease the expensive permanent magnet material by almost one-fifth. Hindawi Publishing Corporation e Scientiﬁc World Journal Volume 2015, Article ID 812149, 6 pages http://dx.doi.org/10.1155/2015/812149 Hindawi Publishing Corporation e Scientiﬁc World Journal Volume 2015, Article ID 812149, 6 pages http://dx.doi.org/10.1155/2015/812149 Research Article On the Stator Slot Geometry of a Cable Wound Generator for Hydrokinetic Energy Conversion Mårten Grabbe, Sandra Eriksson, and Mats Leijon The Swedish Centre for Renewable Electric Energy Conversion, Division of Electricity, The ˚Angstr¨om Laboratory, Uppsala University, P.O. Box 534, 751 21 Uppsala, Sweden 2. Reference Machine Characteristics The 5 kW reference machine has 120 poles and is designed for the low velocities presented by tidal currents. Surface mounted Nd2Fe14B magnets are used and the stator is stacked with laser cut M800-100A sheets with a single row of six 16 mm2 cables in each slot as seen in Figure 1.h The magnet width and slot opening design of the refer- ence machine have largely evolved from a previous study of 2 The Scientific World Journal 2 Table 3: Reference machine performance at its nominal operating point compared with the suggested improved design. Parameter Reference New design Efficiency 86.4% 87.0% B in stator tooth 1.69 T 1.57 T Hysteresis losses in teeth 0.149 kW 0.128 kW Eddy current losses in teeth 0.037 kW 0.032 kW Total losses 0.785 kW 0.748 kW Load angle 7.5∘ 6.2∘ Stator weight 598 kg 532 kg Stator Tooth tip Air gap wslot wwaist wopening dc-a dc-c dc-s Figure 2: A stator slot opening and the two cables closest to the air gap showing the geometry parameters used in this study. PM Rotor ring Air gap Yoke Slot opening Cable Stator tooth Waist Figure 1: The geometry used in the FE model of the reference machine. Table 3: Reference machine performance at its nominal operating point compared with the suggested improved design. Parameter Reference New design Efficiency 86.4% 87.0% B in stator tooth 1.69 T 1.57 T Hysteresis losses in teeth 0.149 kW 0.128 kW Eddy current losses in teeth 0.037 kW 0.032 kW Total losses 0.785 kW 0.748 kW Load angle 7.5∘ 6.2∘ Stator weight 598 kg 532 kg Figure 1: The geometry used in the FE model of the reference machine. Stator Tooth tip Air gap wslot wwaist wopening dc-a dc-c dc-s Figure 2: A stator slot opening and the two cables closest to the air gap showing the geometry parameters used in this study. Stator Table 1: Reference machine main design parameters. Power 5 kW Voltage 150 V Rotational speed 10 rpm Number of poles 120 Fractional winding 7/5 Stator outer diameter 2000 mm Stator inner diameter 1835 mm Air gap 10.5 mm Machine length 270 mm Magnet height 13 mm Magnet width 32 mm PM remanence 1.22 T Table 2: Stator slot geometry of the reference machine compared to the suggested improved geometry. Table 2: Stator slot geometry of the reference machine compared to the suggested improved geometry. 2. Reference Machine Characteristics Figure 2: A stator slot opening and the two cables closest to the air gap showing the geometry parameters used in this study. Parameter Reference New design Slot opening 4 mm 4 mm Slot width (𝑤slot) 8 mm 7.6 mm Cable diameter 7 mm 7 mm Waist width (𝑤waist) 6 mm 6.1 mm Waist factor 0.75 0.8 Cable to cable (𝑑𝑐–𝑐) 2 mm 0.8 mm Cable to air gap (𝑑𝑐–𝑎) 5.5 mm 1.3 mm Cable to stator (𝑑𝑐–𝑠) 0.5 mm 0.3 mm Slot depth 58 mm 47.6 mm The main characteristics of the reference generator are presented in Table 1. The stator slot geometry is detailed in column one of Table 2 and Figure 2, and the resulting performance is summarized in the first column of Table 3. 4. Simulations of the Stator Slot Geometry The parameters that are changed are the the waist (𝑤waist), the distance between cables (𝑑𝑐−𝑐), the distance between cable and air gap (𝑑𝑐−𝑎), and the distance between cable and stator (𝑑𝑐−𝑠), as shown in Figure 2. Load angle (deg) Simulations are performed using the model presented in Section 3. One parameter at a time is changed, while all other parameters are fixed to their reference value. All computations are performed at nominal load and nominal speed. If the slot depth is changed, the stator outer diameter is adjusted accordingly to maintain the same yoke width. As the voltage and power are kept fixed, changes in the axial length are allowed. The changes are a few millimetres at most and are only presented indirectly through small changes in copper losses, iron losses, and stator weight. Waist factor Waist factor The generator is intended to be used with a diode rectifier. In the simulations, the generator is connected to a purely resistive load, which corresponds to a power factor of unity as is the case with diode rectification. During the analysis, the voltage and power are kept constant. According to the circuit theory, the load angle, 𝛿, for a generator with unity power factor can be calculated as sin(𝛿) = 𝑋𝐼/𝐸, where 𝑋is the synchronous reactance, 𝐼is the current, and 𝐸is the internal voltage. The change in load angle can therefore be seen as a direct measurement of change in the machine reactance. Hysteresis loss Load angle Figure 3: The hysteresis loss in the stator teeth and the load angle as a function of the waist factor. 85 85.5 86 86.5 87 Efficiency (%) 1 2 3 4 5 6 7 7.5 8 8.5 9 Load angle (deg) Efficiency Load angle dc-c (mm) Figure 4: The efficiency and the load angle as a function of the distance between cables. 85 85.5 86 86.5 87 Efficiency (%) 1 2 3 4 5 6 7 7.5 8 8.5 9 Load angle (deg) d ( ) Load angle (deg) Efficiency (%) 4.1. Waist Design. In the reference case, the slot width is 8 mm and the waist is 6 mm at its narrowest point. The waist factor, defined as 𝑤waist/𝑤slot, is thus 0.75 in the reference geometry. A pronounced waist keeps the cable firmly in place and reduces the area in the tooth with high magnetic induction, thereby slightly reducing the iron losses. 3. FE Model of the Generator The generator is designed with the aid of an in-house developed design tool and the combined set of field and circuit equations are solved in the finite element environment ACE [11]. The magnetic field inside the core of the generator is assumed to be axisymmetrical and modeled in two dimen- sions. The displacement field is neglected and the permanent magnets are modeled using the current sheet approach [12]. Furthermore, coil end impedances are introduced in the circuit equations, the laminated stator core is modeled using a singl-valued magnetization curve, and a correction factor of 1.5 is used for all iron losses.h a cable wound linear generator [8]. In this case, however, the focus is on the interior geometry of the stator slots.i At first, it should be noted that the cable windings are not inserted radially from the air gap. Rather, the cable is inserted axially into each stator slot position and wound in a fractional (7/5) wave winding. No slot wedges are used, but instead the stator slot opening is narrow to prevent the cables from entering the air gap. Furthermore, a small waist is introduced between each cable to prevent them from rubbing against each other during the winding procedure and to keep them fixed in place during operation. The model used is described in more detail in [13] and simulations of the first prototype have been compared to experiments in [10]. The mesh includes around 33000 elements and second-order shape functions are used. The Scientific World Journal 3 Hysteresis loss in teeth (kW) Waist factor 7 8 9 10 Load angle (deg) Hysteresis loss Load angle 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 0.14 0.15 0.16 0.17 Figure 3: The hysteresis loss in the stator teeth and the load angle as a function of the waist factor. Hysteresis loss in teeth (kW) Waist factor 7 8 9 10 Load angle (deg) 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 0.14 0.15 0.16 0.17 5. Results and Discussion Incorporating all the small changes in the geometry discussed above may add up to a significant improvement. However, none of the predicted improvements would be worthwhile if it turned out to complicate the manufacturing and the assembly procedure. Winding the reference machine went smoothly, most likely thanks to proper stacking and good alignment of the stator sheets. The result from the reference machine has validated the results of the simulations as presented in [10]. This experience gave confidence to incorporate some changes in the second prototype [15]. There, the winding was moved closer to the air gap (2.4 mm instead of 5.5 mm), the cables were moved closer to each other (1 mm instead of 2 mm), and the gap between cable and stator was decreased to 0.4 mm. Again, the winding procedure went smoothly without appreciable wear on the cable insulation. Figure 5: The change in weight and the load angle as a function of the distance from the winding to the air gap. 85.5 86 86.5 87 Efficiency (%) 1 1.5 2 2.5 Efficiency 0.2 0.1 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 dc-s (mm) B in tooth at load (T) B in tooth Figure 6: Efficiency and B-field in tooth at nominal load for varying distance between cable and stator. 85.5 86 86.5 87 Efficiency (%) 1 1.5 2 2.5 0.2 0.1 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 d (mm) B in tooth at load (T) Experience from winding the second prototype has given confidence that further incremental changes to the geometry may be implemented in practice. A tighter gap between cable and stator (0.3 mm) would render a less pronounced waist possible. Experience also indicate that the cables could be moved closer to the air gap (1.3 mm), further decreasing the slot depth and the generator outer diameter. The new stator slot design is shown in Figure 7 and compared to the reference machine in Tables 2 and 3. The proposed changes would, according to simulations, improve the efficiency from 86.4% to 87.0% and lower the load angle from 7.5∘to 6.2∘. 4. Simulations of the Stator Slot Geometry However, the waist also increases the leakage flux resulting in a higher load angle. As can be seen in Figure 3, no waist decreases the load angle but increases the hysteresis losses in the stator tooth. Without a waist, however, some other means of protecting the cables from rubbing against each other during winding would have to be implemented. Figure 4: The efficiency and the load angle as a function of the distance between cables. 4.2. Cable to Cable. The distance between cables (𝑑𝑐−𝑐) may have practical considerations during the winding procedure. Keeping the cables close to each other results in shorter stator teeth and a more compact design. However, if the cables are positioned too closely, a more pronounced waist may be required to prevent them from rubbing against each other during winding. The coil end positioning may also be affected by the distance between cables. A distance of 2 mm was used successfully in the reference machine, making a more compact design look viable. 4.3. Cable to Air Gap. Moving the winding closer to the air gap is perhaps the most straight forward way to achieve a more compact design. One might have to consider, depend- ing on winding scheme, if the coil ends need a certain space to the air gap. A distance of 5.5 mm from the air gap to the first cable position was chosen in the reference machine. It turned out to work well in practice, as the coil ends could be pushed backwards away from the air gap, leaving room for improvements in this area as well.hffi Gains in both efficiency and load angle can be achieved by moving the cables closer to each other (see Figure 4). Perhaps more importantly, the stator weight and stator outer diameter can be reduced by 4% and 8 mm, respectively. It should be noted though, that the same space would still be required for the coil ends. The distance to the air gap hardly affects the efficiency at nominal load conditions. However, as can be seen in Figure 5, the weight of the machine and load angle are both decreased as the windings are moved closer to the air gap. 5. Results and Discussion Perhaps more significantly, the stator weight would be decreased by 11%, and the outer diameter would be decreased by 20.4 mm allowing for further savings on the support structure.h 5 B in tooth at load (T) Efficiency (%) The decrease in load angle can be translated into either a decrease in magnetic material necessary to maintain the nominal design point of 150 V and 5 kW at a load angle of 7.5∘, or an increase in power output at the same voltage and load angle as the reference machine. Simulations predict that the new design can reach the nominal design point utilizing 19% less permanent magnet material, or a 22% increase in electrical power output at the reference voltage and load angle. Figure 6: Efficiency and B-field in tooth at nominal load for varying distance between cable and stator. 4.4. Slot Width. The distance between the cable and the stator (𝑑𝑐−𝑠) might be the single parameter that most clearly affects the winding procedure. Assuming the stator sheets are well aligned and properly stacked, a certain distance between the cable and the stator is still needed to allow for a smooth and easy winding procedure. A too tight design might cause unnecessary wear on the cables. 4. Simulations of the Stator Slot Geometry 4 The Scientific World Journal The Scientific World Journal The Scientific World Journal 4 90 100 110 Weight (%) 1 2 3 4 5 6 7 8 9 10 11 6 8 10 Load angle (deg) Weight Load angle dc-a (mm) Figure 5: The change in weight and the load angle as a function of the distance from the winding to the air gap. 90 100 110 Weight (%) 1 2 3 4 5 6 7 8 9 10 11 6 8 10 Load angle (deg) dc-a (mm) The slot width is changed, which results in a new tooth width. Thus, clear changes are seen in the B-field in the stator tooth as 𝑑𝑐−𝑠is changed (see Figure 6). In other words, if a larger gap between cable and slot is needed, the design will also have to incorporate a slightly larger stator diameter to accommodate sufficiently wide stator teeth. Load angle (deg) Weight (%) Weight (%) Acknowledgments The work reported was financially supported by the Swedish Centre for Renewable Electric Energy Conversion, STandUP for Energy and Swedish Research Council (Grant no. 621- 2009-4946). Construction of the prototype generators has been supported by Vattenfall AB, the Swedish Energy Agency, ˚Angpannef¨oreningen’s Foundation for Research and Devel- opment (˚AForsk), and the J. Gust. Richert Foundation. The electrical design of the first prototype performed by Karin Thomas is deeply appreciated. [7] S. Eriksson, H. Bernhoff, and M. Leijon, “A 225 kW direct driven PM generator adapted to a vertical axis wind turbine,” Advances in Power Electronics, vol. 2011, Article ID 239061, 7 pages, 2011. [8] O. Danielsson, M. Leijon, and E. Sj¨ostedt, “Detailed study of the magnetic circuit in a longitudinal flux permanent-magnet synchronous linear generator,” IEEE Transactions on Magnetics, vol. 41, no. 9, pp. 2490–2494, 2005. [9] S. Alfredsson, B. Hernn¨as, and H. Bergstr¨om, “Assembly of generators with rated voltage higher than 100 kV,” in Proceedings of the International Conference on Power System Technology (PowerCon ’00), vol. 1, pp. 189–193, 2000. 6. Conclusions Cables with a diameter of 7 mm were used to wind the reference machine with a slot width of 8 mm (i.e., 𝑑𝑐−𝑠= 0.5 mm). The winding procedure went smoothly. It should however be noted that the prototype was wound by hand, and the distance between cable and stator might have to be reevaluated when utilizing industrial robots for automated winding [14]. The finite element analysis of the cable wound generator shows that the performance can be improved by apparently small changes in the stator slot geometry. A new design is proposed based on practical experience from winding two prototypes, and simulations predict an increase in efficiency of 0.6 percentage points, while the stator core weight is The Scientific World Journal 5 (a) 1.9 1.71 1.52 1.33 1.14 0.95 0.76 0.57 0.38 0.19 0 (b) Figure 7: Illustration of the magnetic field distribution in the reference machine (to the left) and the new design (to the right). 1.9 1.71 1.52 1.33 1.14 0.95 0.76 0.57 0.38 0.19 0 (b) the reference machine (to the left) and the new design (to the right) 1.9 1.71 1.52 1.33 1.14 0.95 0.76 0.57 0.38 0.19 0 (b) (a) (b) Figure 7: Illustration of the magnetic field distribution in the reference machine (to the left) and the new design (to the right). of the 7th European Wave and Tidal Energy Conference (EWTEC ’07), Porto, Portugal, 2007. reduced by 11% and the load angle is decreased by 17%. The more compact design allows for further savings on the support structure. The decrease in load angle can be translated into a 19% decrease in magnetic material at the nominal design point. Finally, it can be concluded that the load angle is influenced by small alterations in the stator geometry. Therefore, a thorough evaluation of the stator slot geometry is important when optimizing a generator design. [3] L. Drouen, J. F. Charpentier, E. Semail, and S. Clenet, “Study of an innovative electrical machine fitted to marine current tur- bines,” in Proceedings of the OCEANS 2007-Europe Conference, pp. 1–6, Aberdeen, Scotland, June 2007. [4] J. Weilepp, “Progress in wave and tidal current technology,” International Journal on Hydropower and Dams, vol. 16, no. 4, pp. 95–100, 2009. Conflict of Interests [5] J. Clarke, G. Connor, A. Grant, C. Johnstone, and S. Ordonez- Sanchez, “Analysis of a single point tensioned mooring system for station keeping of a contra-rotating marine current turbine,” IET Renewable Power Generation, vol. 4, no. 6, pp. 473–487, 2010. The authors declare that there is no conflict of interests regarding the publication of this paper. [6] O. Keysan, A. S. McDonald, and M. Mueller, “A direct drive permanent magnet generator design for a tidal current turbine (SeaGen),” in Proceedings of the IEEE International Electric Machines and Drives Conference (IEMDC ’11), pp. 224–229, May 2011. References Submit your manuscripts at http://www.hindawi.com Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Structures Journal of International Journal of Rotating Machinery Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Industrial Engineering Journal of Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Tribology Advances in Energy Journal of Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Hindawi Publishing Corporation http://www.hindawi.com Journal of Engineering Volume 2014 Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 International Journal of Photoenergy Nuclear Installations Science and Technology of Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Solar Energy Journal of Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Wind Energy Journal of Power Electronics Advances in Fuels Journal of Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Nuclear Energy International Journal of Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 High Energy Physics Advances in Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Mechanical Engineering Advances in Journal of Petroleum Engineering Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 The Scientific World Journal Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Journal of Renewable Energy Combustion Journal of Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Submit your manuscripts at http://www.hindawi.com International Journal of Rotating Machinery Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Industrial Engineering Journal of Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Energy Journal of Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Solar Energy Journal of Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Wind Energy Journal of Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Mechanical Engineering Advances in Journal of Petroleum Engineering Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 The Scientific World Journal Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 International Journal of Rotating Machinery Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Wind Energy Journal of Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Mechanical Engineering Advances in The Scientific World Journal Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 International Journal of Rotating Machinery The Scientific World Journal Hi d i P bli hi C i Petroleum Engineering Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 References [1] K. 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Saito, “Finite element solution of permanent magnetic field,” IEEE Transactions on Magnetics, vol. 26, no. 2, pp. 383–386, 1990. [13] S. Eriksson, Direct Driven Generators for Vertical Axis Wind Turbines, Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, 2008. [14] E. Hultman and M. Leijon, “Utilizing cable winding and industrial robots to facilitate the manufacturing of electric machines,” Robotics and Computer-Integrated Manufacturing, vol. 29, no. 1, pp. 246–256, 2013. [15] K. Yuen, S. Lundin, M. Grabbe, E. Lalander, A. Goude, and M. Leijon, “The S¨oderfors project: construction of an experimental hydrokinetic power station,” in Proceedings of the 9th European Wave and Tidal Energy Conference (EWTEC '11), pp. 1–5, Southampton, UK, 2011. 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W2057155863.txt	https://www.ocl-journal.org/articles/ocl/pdf/2002/04/ocl200294p218.pdf	fr		PRESENTATION Des acides gras poly-insaturés au mélange harmonieux alphalinolénique, linoléique, oléique. Un chemin malaisé, mais plein d’avenir	Oilseeds and fats, crops and lipids/OCL. Oilseeds & fats crops and lipids	2,002	cc-by	1,224	PRESENTATION Des acides gras poly-insaturés au mélange harmonieux alphalinolénique, linoléique, oléique. Un chemin malaisé, mais plein d'avenir Oléagineux, Corps Gras, Lipides. Volume 9, Numéro 4, 218-9, Juillet - Août 2002, Dossier : Equilibre entre acides gras insaturés : contributions à l'étude de la prévention des maladies cardio-vasculaires Auteur(s) : François MENDY, 2, rue du Calvaire 92210 Saint-Cloud. ARTICLE La théorie lipidique de l'athérosclérose prit son plein essor dans les années 1960. Fondée sur des données d'épidémiologie rétrospective (A. Keys, F. Grande, 1957, A. Keys et D.M. Hegsted, 1965), elle mettait en avant 3 facteurs : le cholestérol total sanguin (critère principal), les acides gras poly-insaturés, les acides gras saturés. D'essence essentiellement pragmatique, elle enjambait un certain nombre de... boîtes noires et consacrait le règne des épidémiologistes-statisticiens pour un demi-siècle. Ce n'est que dans les années 1990 qu'apparut la nécessité de la mise en évidence d'un mécanisme d'action pour revendiquer une action sur une fonction, et s'insérer réellement dans l'evidence-based medecine. Quoi qu'il en soit pour de longues années, le rapport polyinsaturés/saturés (P/S) allait devenir le dogme séparant le monde du bien et le monde du mal. Et très vite en France, l'amalgame poly- insaturés = linoléique + matières grasses végétales, saturés = matières grasses animales, se fit. Pendant cette période, quatre grands courants de pensée biochimiste accueillaient avec une certaine réticence cette simplification qui semblait quelque peu abusive, mais qui, malgré tout, dans l'ensemble, permettait d'améliorer leurs moyens d'étude. * Cela faisait déjà deux siècles que des biochimistes se posaient le problème de la régulation de la synthèse du cholestérol, de son métabolisme, de son rôle au niveau des membranes et de la synthèse des hormones stéroïdiennes. La découverte des inhibiteurs de la synthèse du cholestérol, les statines, amena la preuve définitive de la validité de la théorie lipidique de l'athérosclérose. Mais en même temps, une question se posait : quel niveau de population mettre sous satines ? D'autant que, lentement, le critère unique de 1965, le taux de cholestérol total plasmatique était remplacé par trois critères : le mauvais cholestérol, C-LDL, le bon cholestérol, C-HDL, et plus lentement les triglycérides. * Dans un autre domaine, depuis près d'un siècle, d'autres biochimistes s'intéressaient soit aux acides gras, soit à des composés cycliques semblant avoir quelques liens de parenté avec les acides gras. Il fallut trente ans pour que la jonction se fît. Article disponible sur le site http://www.ocl-journal.org ou http://dx.doi.org/10.1051/ocl.2002.0218 * Une longue série de chercheurs (G.O. Burr, Holman, J. Mead, H. Sprecher, W.E.M. Lands...) fit exploser la notion de « poly-insaturés = linoléique » en trois familles de poly-insaturés, la famille des acides gras en n-6, avec comme acide gras parent l'acide linoléique non synthétisable et la famille des acides gras en n-3 avec comme acide gras parent l'acide alphalinolénique non synthétisable et la famille en n-9 avec comme acide gras parent l'acide oléique, un acide gras mono-insaturé, synthétisable sous conditions. En mars 1982, R.T. Holman montrait de façon définitive que l'acide alphalinolénique par voie intraveineuse guérissait le syndrome neurologique déclenché par sa carence, établissait un besoin minimal par voie veineuse de 44 mg d'acide linolénique par kg, soit 0,54 % des calories totales (ce qui donne par voie orale 0,82 % si l'ALA est pour 66 % en sn-2). Le rapport LA/ALA était de 6,1. En même temps, la voie des prostaglandines, ouverte dès les années 1930 par U.S. von Euler, explosait également, aboutissant à un 2e prix Nobel en 1982, avec S. Bergstrom, B. Samuelson et J. Vane. Très rapidement, ce domaine était encore enrichi par la découverte par P. Borgeat des leucotriènes et autres dérivés oxigènes biologiques des acides gras poly-insaturés ou hautement insaturés. L'existence d'effets opposés de dérivés oxygénés, thromboxane, prostacycline, provenant selon des régulations complexes mais naturellement, d'un même acide gras. Hautement insaturé de la famille en n-6 du linoléique, l'acide arachidonique fait ainsi passer les acides gras poly-insaturés du rôle d'ange à celui de démon, selon leur niveau d'apport, selon le rapport LA/ALA. Et pour compléter ce bref résumé d'une longue histoire, une réflexion de W.E.M. Lands mérite d'être citée : « It was not clear at the time (or even today !) what the saturated fat might actually be doing to harm health » (Inform, avril 2002, 13, 346). De 1975 à 1994 une série d'études soutenues par la filière des oléagineux , conduites dans les unités de B. Jacotot et S. Renaud amenaient à mieux comprendre le rapport acide linoléique/acide alphalinolénique en sn-2 et ses implications en médecine humaine. Cette période se termina par la publication de la Lyon Heart Diet Study (Lancet, 1994, 343, 1454-59). Dans cette étude, au niveau du lot expérimental, l'apport en LA était de 3,6 % de l'énergie totale (ET), en ALA de 0,81 % de l'ET (dont 66 % en sn-2), le rapport LA/ALA de 4,44/1, cela pour 1 928 calories. L'apport en oléique était de 12,9 % de l'ET. Selon l'expression de W.E.M. Lands, il existe quelques différences « between the discipline of science and the marketing of science ». Le monde de la communication en retint qu'il fallait augmenter la consommation d'huile d'olive, et le Comité des ANC 2000, que l'on pouvait recommander un apport de 20 % de l'ET sous forme d'oléique. Il apparut en 1994 que l'on disposait de très peu d'éléments sur les limites intéressantes, la flexibilité possible des apports en oléique, et secondairement même des apports en linoléique. Plusieurs auteurs préconisaient en effet des taux plus élevés que ceux de la Lyon Heart Diet Study, au moins de 5 à 6 % de l'ET sous forme d'acide linoléique. Il fut décidé de conduire cette étude préliminaire en milieu fermé, chez les moines de l'abbaye de Solesmes. Ce sont des éléments de cette étude qui sont rapportés dans ce numéro. Mais quels critères choisir, puisque ce ne pouvait être celui de la réduction de mortalité comme dans la Lyon Diet Heart Study. Quels pouvaient être les meilleurs critères discriminants corrélables avec l'apport de mélanges particuliers de triglycérides alimentaires ? Le moment est venu de parler brièvement de ce quatrième courant de la pensée biochimique, lié aux relations entre les protéines et les lipides. Les cénapses lipidoprotéiques de Machebœuf remontent à plus d'un demi-siècle, elles ont pourtant ouvert la voie aux lipoprotéines, et finalement attiré l'attention sur la partie protéique des lipoprotéines, les apoprotéines. D'elles dépendent le transport des lipides, les interactions lipoprotéines-récepteurs, la régulation de l'activité des enzymes impliquées dans le métabolisme des particules lipoprotéiques. Le prix Nobel de Brown et Goldstein a définitivement fait comprendre le rôle du récepteur LDL et de l'ApoB dans l'une des phases de la capture du cholestérol, mais le mérite de la première vision synthétique du flux des particules lipoprotéiques des apoprotéines, revient à P. Alaupovic, rapidement suivi en France par J.C. Fruchart. Il s'avère de plus en plus que ces particules lipoprotéiques, ces apoprotéines, peuvent être parmi les meilleurs facteurs discriminants, les meilleurs marqueurs biologiques, pour le type d'étude que nous voulions poursuivre. C'est pour cette raison que nous avons demandé à P. Alaupovic de nous faire l'honneur d'ouvrir ce numéro spécial. Et sans doute est-il intéressant d'attirer l'attention sur le fait que ces notions, qui peuvent paraître difficiles, ces dosages qui peuvent paraître complexes ouvrent la voie et valident des marqueurs utilisables dès maintenant par le médecin praticien comme le simple rapport cholestérol non HDL/cholestérol HDL.
https://openalex.org/W2036468797	https://hal-univ-evry.archives-ouvertes.fr/hal-02489938/document	English	null	Serum 1H-NMR Metabolomic Fingerprints of Acute-On-Chronic Liver Failure in Intensive Care Unit Patients with Alcoholic Cirrhosis	PloS one	2,014	cc-by	7,697	To cite this version: Roland Amathieu, Mohamed N Triba, Pierre Nahon, Nadine Bouchemal, Walid Kamoun, et al.. Serum 1H-NMR metabolomic fingerprints of acute-on-chronic liver failure in intensive care unit patients with alcoholic cirrhosis. PLoS ONE, 2014, 9 (2), pp.e89230. 10.1371/journal.pone.0089230. hal-02489938 Serum 1H-NMR metabolomic fingerprints of acute-on-chronic liver failure in intensive care unit patients with alcoholic cirrhosis Roland Amathieu, Mohamed N Triba, Pierre Nahon, Nadine Bouchemal, Walid Kamoun, Hakim Haouache, Jean-Claude Trinchet, Philippe Savarin, Laurence Le Moyec, Gilles Dhonneur HAL Id: hal-02489938 https://univ-evry.hal.science/hal-02489938v1 Submitted on 27 Feb 2020 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Abstract These authors contributed equally to this work. function, multiple-organ failure, and death within a few days or weeks. These patients with cirrhosis need ICU management. Serum 1H-NMR Metabolomic Fingerprints of Acute-On- Chronic Liver Failure in Intensive Care Unit Patients with Alcoholic Cirrhosis Roland Amathieu1,2., Mohamed N. Triba2., Pierre Nahon3, Nadia Bouchemal2, Walid Kamoun1, Hakim Haouache1, Jean-Claude Trinchet3, Philippe Savarin2, Laurence Le Moyec4, Gilles Dhonneur1 1 Service d’Anesthe´sie et des Re´animations Chirurgicales, Universite´ Paris 12, Hoˆpital Henri Mondor, Assistance Publique des Hoˆpitaux de Paris (AP-HP), Cre´teil, France, 2 Universite Paris 13, Sorbonne Paris Cite´, Laboratoire Chimie, Structures, Proprie´te´s de Biomate´riaux et d’Agents The´rapeutiques (CSPBAT), Unite´ Mixte de Recherche (UMR) 7244, Centre National de Recherche Scientifique (CNRS), Equipe Spectroscopie des Biomole´cules et des Milieux Biologiques (SBMB), Bobigny, France, 3 Service d’He´patologie et Universite´ Paris 13, Hoˆpital Jean Verdier, Assistance Publique des Hoˆpitaux de Paris (AP-HP), Bondy, France, 4 Institut National de la Sante´ et de la Recherche Me´dicale (INSERM) Unite´ de Biologie Inte´grative des Adaptations a` l’Exercice (UBIAE U902), Universite´ d’Evry, Evry, France Abstract Introduction: Acute-on-chronic liver failure is characterized by acute deterioration of liver function in patients with compensated or decompensated, but stable, cirrhosis. However, there is no accurate definition of acute-on-chronic liver failure and physicians often use this term to describe different clinical entities. Metabolomics investigates metabolic changes in biological systems and identifies the biomarkers or metabolic profiles. Our study assessed the metabolomic profile of serum using proton nuclear magnetic resonance (1H-NMR) spectroscopy to identify metabolic changes related to acute-on-chronic liver failure. Patients: Ninety-three patients with compensated or decompensated cirrhosis (CLF group) but stable liver function and 30 patients with cirrhosis and hospitalized for the management of an acute event who may be responsible of acute-on-chronic liver failure (ACLF group), were fully analyzed. Blood samples were drawn at admission, and sera were separated and stored at –80uC until 1H-NMR spectral analysis. Using orthogonal projection to latent-structure discriminant analyses, various metabolites contribute to the complete separation between these both groups. Results: The predictability of the model was 0.73 (Q2Y) and the explained variance was 0.63 (R2Y). The main metabolites that had increased signals related to acute-on-chronic liver failure were lactate, pyruvate, ketone bodies, glutamine, phenylalanine, tyrosine, and creatinine. High-density lipids were lower in the ALCF group than in CLF group. Conclusion: A serum metabolite fingerprint for acute-on-chronic liver failure, obtained with 1H-NMR, was identified. Metabolomic profiling may aid clinical evaluation of patients with cirrhosis admitted into intensive care units with acute-on- chronic liver failure, and provide new insights into the metabolic processes involved in acute impairment of hepatic function. Editor: Petras Dzeja, Mayo Clinic, United States of America Editor: Petras Dzeja, Mayo Clinic, United States of America Received October 18, 2013; Accepted January 17, 2014; Published February 19, 2014 Copyright: 2014 Amathieu 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. athieu 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. Funding: The authors have no support or funding to report. Competing Interests: The authors have declared that no competing interests exist. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. . Metabolomic Approach of ACLF Other studies show that metabolomics can fingerprint the differences between compensat- ed and decompensated cirrhosis, and between cirrhosis caused by alcohol or viruses [10,11]. We have previously shown that a metabolomic approach was a powerful tool in assessing the severity of chronic liver failure in alcohol-induced cirrhosis within a cohort of patients without ACLF. In our previous study, the severity of chronic liver failure was evaluated using the MELD score, and correlated well with impairment of lipid, glucose, and amino acid metabolism [6]. The date of inclusion into the study was the date when blood was collected. Blood samples were drawn under fasting conditions for the CLF group and immediately at admission for the ALCF group. Serum was separated and stored at –80uC until further analyses. Gender, age, the presence of ascites or hepatic encephalopathy, serum bilirubin, albumin and prothrombin levels, the International Normalized Ratio, serum alanine-aminotrans- ferase (ALT) activity, serum aspartate-aminotransferase (AST) activity, creatininemia, and platelet counts, were recorded at inclusion. For patients with ALCF, diagnosis was made as they entered the ICU ward. The main diagnoses were sepsis, GIB, acute renal failure, or hepatic encephalopathy. Gastrointestinal bleeding was defined as any hematemesis or melena with transfusion of two or more units of blood. Sepsis was defined as proposed in the International Survey Sepsis Campaign [15]. Blood lactate, hemodynamic parameters, and the use of catecholamine and mechanical ventilation were recorded. To evaluate the severity of the disease, the Sequential Organ Failure Assessment (SOFA) score was calculated during the first day of admission [16]. For all patients, the severity of chronic liver failure was calculated using both the Child–Pugh–Turcott and MELD scores [17,18]. For the ACLF group, the more recent Child–Pugh–Turcott score before hospitalization in the ICU was taken into account. In ICU investigations of liver disease, some studies have evaluated the different metabolic profiles after transplantation and have compared non-survivor to survivor or graft-failure cases: they have shown improvement in patients’ liver status after transplantation [12,13]. Another study that assessed sera using 1H- NMR showed the potential of the metabolomic profile to evaluate patients with fulminant hepatic failure and to predict an unfavorable outcome [14]. However, none of these studies have focused on the changes in metabolism that occur in patients with cirrhosis and ACLF. Metabolomic Approach of ACLF EASL-CLIF consortium definitions were not used to include patient in ACLF group. Compensated and decompensated patients who had stable cirrhosis, and who had been referred to the hepatology unit, within the same period, for either therapeutic management (paracenthesis) or a screening procedure were included in the CLF group. EASL-CLIF consortium definitions were not used to include patient in ACLF group. Compensated and decompensated patients who had stable cirrhosis, and who had been referred to the hepatology unit, within the same period, for either therapeutic management (paracenthesis) or a screening procedure were included in the CLF group. after onset, to an initiating event that is accepted as jaundice, or to any other symptom that pertains to hepatic dysfunction. In the EASL-CLIF consensus, patients with acute decompensated cirrhosis were classified in three grades according to renal impairment and/or others extra-hepatic organ failures. A better definition of ACLF could be obtained using new biomarkers. Nevertheless, it is now widely accepted that the search for a single biomarker that can be used in routine clinical practice to diagnosis patients with ACLF is probably unrealistic. Cirrhosis was defined by a liver biopsy or the presence of at least two compatible diagnostic factors from the following: radiology (ultrasound, computed tomography, or magnetic-resonance imag- ing), clinical examinations (at least two indicators: hepatomegaly, splenomegaly, encephalopathy, at least two arterial spiders, collateral venous circulation, or jaundice), and/or biological factors (increased bilirubinemia, a prolonged International Nor- malized Ratio, or thrombocytopenia). Metabolomics, which is the study of metabolic changes in an integrated biological system using multiparametric analyses, may help identify biomarkers that characterize the metabolic profiles of a disease, and/or evaluate metabolic modifications after treatment has been initiated [5]. Metabolomics, using proton nuclear magnetic resonance (1H-NMR) spectroscopy, when applied to liver disease, has shown a close relationship between metabolic abnormalities and the severity of the disease in sera and tissues [6,7,8]. Only patients with alcoholic cirrhosis were considered. Exclu- sion criteria were infection with the human immunodeficiency virus, hepatitis B or C viruses, evidence of hepatocarcinoma (as judged by ultrasonographic findings and serum a-fetoprotein concentration .50 ng/mL), and a past history of acute decom- pensation during the previous 6 months. Jimenez et al. have shown that using 1H-NMR to assess the metabolomics of sera from patients with cirrhosis could discrim- inate between patients with minimal hepatic encephalopathy and those with no encephalopathy [9]. Materials and Methods Patients and Collection of Sera Samples Patients and Collection of Sera Samples Between August 2010 and December 2010, all consecutive patients with cirrhosis and referred to our Liver Center were screened for the study. Unstable cirrhotic patients with high risk of liver and/or other organ failure secondary to a severe acute event, mainly gastrointestinal bleeding (GIB) or sepsis, hospitalized and managed in ICU were included in the ACLF group. Asian or Ethics Statement The Institutional Review Board of Jean Verdier University Hospital approved the protocol and the French Research Delegation Office accepted the creation of a dedicated bio- collection for the patients included in the protocol. The CNIL (National Informatics and Liberty Commission) also approved the creation of both bio-collection and database. Depending of the site of hospitalization (ward or ICU) and the mental status of the patient, the written consent was obtained directly from the patient at inclusion or from the Person of Confidence designed by the patient (pre-emptively) or by the family relatives. In case, the patient survived, secondary written consent from the patient was systematically seek. Metabolomic Approach of ACLF We hypothesize that patients with cirrhosis and hospitalized in ICUs have acute changes to their metabolism that can be identified using 1H-NMR to evaluate their sera. These metabolic changes could then identify patients with ACLF, independently of trigger events. Thus, the purpose of our study was to assess whether the metabolomic profiles of sera, obtained by 1H-NMR spectroscopy, were modified in patients with alcohol-induced cirrhosis and hospitalized in an ICU for an acute event known to cause ACLF, when compared to patients with compensated or decompensated, but stable, cirrhosis. Another aim was to identify the impaired common metabolic pathways. Consequently, the decrease or the increase of the different metabolic pathways will be discussed. Introduction Acute-on-chronic liver failure (ACLF) is a syndrome character- ized by acute deterioration of liver function in patients with compensated or decompensated, though stable, cirrhosis. ACLF is a serious condition that has varied etiologies and manifestations, as well as a high mortality rate [1]. ACLF commonly occurs after an acute event, and is often associated with extra-hepatic organ failure. The most common triggers for ACLF include sepsis and digestive bleeding [2]. Any patient with cirrhosis is exposed to acute complications, which, in turn, may occasionally initiate a cascade of events that can lead to further deterioration of liver There is no consistent or clear definition of ACLF in the literature, and physicians often use this term to describe different conditions. Previous studies on ACLF have used different definitions, and there is no overall consensus for the liver failure that occurs, for the causative acute event, or for the diagnosis of the underlying chronic liver disease. Recently, the Asia-Pacific Association for the study of liver disease and the EASL-CLIF consortium have proposed different consensus definition for ACLF [3,4]. In the Asiatic consensus, criteria for defining ACLF vary widely from the level of bilirubin, to the time until deterioration February 2014 \| Volume 9 \| Issue 2 \| e89230 February 2014 \| Volume 9 \| Issue 2 \| e89230 1 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org Metabolomic Approach of ACLF 1H-NMR Spectroscopy For NMR analysis, samples were thawed at room temperature. A volume of 0.6 mL of serum was placed into a 5-mm-diameter tube together with 0.1 mL of D2O, which contained a known amount of fumaric acid. The proton spectra were acquired at February 2014 \| Volume 9 \| Issue 2 \| e89230 February 2014 \| Volume 9 \| Issue 2 \| e89230 PLOS ONE \| www.plosone.org 2 Metabolomic Approach of ACLF 500 MHz on a Varian Unity InovaH spectrometer at 25uC. The signal was acquired after a 90u pulse on 32-K data points for a spectral window of 5000 Hz. The relaxation delay was 4 s. The water signal was suppressed by a pre-saturation sequence using low-power irradiation (0.03 W for 2 s) on the water-signal frequency during the relaxation delay. The Carr-Purcell-Mei- boom-Gill (CPMG) sequence is frequently used by others [9] to suppress the broad protein signal according to its short T2 relaxation time. Because lipids content in the sera of patients with cirrhosis [6] are an important discriminant variable, a single pulse sequence was preferred to preserve the complete lipid profile of the spectra. The resulting free induction decays obtained with 128 transients were processed by MestrecH software. 500 MHz on a Varian Unity InovaH spectrometer at 25uC. The signal was acquired after a 90u pulse on 32-K data points for a spectral window of 5000 Hz. The relaxation delay was 4 s. The water signal was suppressed by a pre-saturation sequence using low-power irradiation (0.03 W for 2 s) on the water-signal frequency during the relaxation delay. The Carr-Purcell-Mei- boom-Gill (CPMG) sequence is frequently used by others [9] to suppress the broad protein signal according to its short T2 relaxation time. Because lipids content in the sera of patients with cirrhosis [6] are an important discriminant variable, a single pulse sequence was preferred to preserve the complete lipid profile of the spectra. The resulting free induction decays obtained with 128 transients were processed by MestrecH software. A score plot illustrated the results. Each point in the score-plot represents the projection of an NMR spectrum (and thus a patient’s sample) on the predictive (horizontal axis) and the first orthogonal component of the model (vertical axis). The loading plot represents the covariance between the Y-response matrix and the signal intensity of the various spectral domains. Multivariate Analyses A principal component analysis (PCA) was first performed to detect any group separation based on NMR-signal variability. This method also enabled detection and exclusion of any outliers, defined as observations located outside the 95% confidence region of the model. Orthogonal projection to latent-structure (OPLS) analysis was run to identify differences between patients in the CLF and ACLF groups. Compared to the classical projection of latent-structure analysis, this method allowed improved interpre- tation of the spectroscopic variations between the groups, by removing information that had no impact on the differences. PCA and OPLS analyses were performed using Simca-P12 (Umetrics, Umea) and in-house MatlabH (Mathworks, Natick, MA) code based on the Trygg and Wold method [19]. Demographic, clinical, and biological features of all patients included in the study are displayed in Table 1, and specific characteristics of the ICU patients included in the OPLS analysis are displayed in Table 2. 1H-NMR Spectroscopy Colors were also used in the loading plot depending of the p-value associated with the correlation between the corresponding bucket intensity and Y variable. The null hypothesis associated with the p-values used in this study was that there was no correlation between X (intensity of the buckets) and Y (CLF or ALCF group) variables. To minimize false-positive rates in multiple comparisons of the 4239 spectral domains, we used the conservative Bonferroni correction, which discards any low significant variables. Thus, for an error rate of 0.01, a metabolite variation in the loading plot was considered significant if its p-value was less 2.4610–6. A Fourier transformation was applied with an exponential window function to produce a 1-Hz broadening line. The spectra were phased and a spline baseline correction was applied with three points at 0.5, 4.6, and 9 ppm. The chemical shifts were referenced using the fumaric-acid signal (6.53 ppm). The spectral region between 0–9 ppm was divided into 9000 spectral regions of 0.001-ppm width, called buckets, using a personal program with RH. Water, urea (signal damaged by water-saturation transfer), and fumaric-acid regions were excluded (1810 variables excluded). A Fourier transformation was applied with an exponential window function to produce a 1-Hz broadening line. The spectra were phased and a spline baseline correction was applied with three points at 0.5, 4.6, and 9 ppm. The chemical shifts were referenced using the fumaric-acid signal (6.53 ppm). The spectral region between 0–9 ppm was divided into 9000 spectral regions of 0.001-ppm width, called buckets, using a personal program with RH. Water, urea (signal damaged by water-saturation transfer), and fumaric-acid regions were excluded (1810 variables excluded). At admission, ICU patients were not fasted whereas patients from the CLF group were. Moreover, some ICU patients were perfused with hydroxyl-ethyl starch, a volume expender, before admission to the ICU. Glucose residues of hydroxyl-ethyl starch produce an intense and broad resonance between 3.35 and 4.06 ppm. For this reason, the region between 3.35 and 4.06 ppm, corresponding to the glucose signal, was excluded from all spectra of the CLF and ACLF groups. Each bucket integral was integrated and scaled to the total summed bucket integrals for each spectrum. Patients’ Characteristics and PCA Analysis Patients’ Characteristics and PCA Analysis A total of 130 patients fulfilled the inclusion criteria: there were 35 patients in the ACLF group and 95 patients in the CLF group. A flow chart of the patients’ characteristics is presented in Figure 1. The data obtained by bucketing the spectra of 130 sera were first analyzed by PCA (not shown). From this analysis, seven outliers were identified: two in the CLF group and five in the ACLF group. The two outliers in the CLF group exhibited high levels of ethanol: 1.11 and 3.66 ppm. Of the five outliers in the ACLF group, three concerned hemorrhagic shock previously transfused. All these outliers were excluded from the OPLS analysis. Identification of Metabolites On the loading plot, positive signals corresponded to the metabolites that had increased concentrations in the sera from patients in the ALCF group. Conversely, a negative signal corresponded to metabolites that had an increased concentration in the sera of patients in the CLF group. At admission, ICU patients were not fasted whereas patients from the CLF group were. Moreover, some ICU patients were perfused with hydroxyl-ethyl starch, a volume expender, before admission to the ICU. Glucose residues of hydroxyl-ethyl starch produce an intense and broad resonance between 3.35 and 4.06 ppm. For this reason, the region between 3.35 and 4.06 ppm, corresponding to the glucose signal, was excluded from all spectra of the CLF and ACLF groups. Each bucket integral was integrated and scaled to the total summed bucket integrals for each spectrum. The buckets were designated according to their central chemical-shift values. Their most probable assignment to a specific metabolite was given according to the spectral assignment and its detailed appearance (multiplet, high-resolution peaks, and broad signals), as previously described in the literature [20,21], and according to our previous signal assignments [6,22]. To help identify the metabolites, statistical analysis of total-correlation spectroscopy was performed (data not shown) [23]. Results Qualitative variables were compared using Fischer’s exact, chi- squared test or the chi-squared trend test with 1 degree of freedom, whereas quantitative variables were compared using the non- parametric Wilcoxon’s test. All reported p values are two-tailed. Associations were considered to be statistically significant at two- tailed a-values of 0.05. OPLS Model and Their Validation Spectra of the sera from the CLF and ACLF groups were identified using the OPLS model, as shown in the score plot in Figure 2A. Examples of typical spectra from each group are shown in Figure 2B and C. The model was built with one predictive and two Y-orthogonal components, and exhibited a good explained variance (R2Y) of 0.73 and a predictability (Q2Y) of 0.63. The goodness-of-fit parameters for the OPLS model, R2Yand Q2Y, were calculated. R2Y represents the explained variance of the Y matrix. Q2Y estimates the predictability of the model. R2Y = 1 indicates perfect description of the data by the model, whereas Q2Y = 1 indicates perfect predictability. For internal validation of the OPLS models, a permutation test (999 permutations) was performed. This evaluated whether the OPLS models, built with the groups, was significantly better than any other OPLS model obtained by randomly permuting the original group attributes. On the score plot, the spectra from the CLF and ACLF groups were well separated along the horizontal axis. Nevertheless, four patients from the ACLF group were misclassified and had similar metabolomic profiles to patients from the CLF group: two patients with ACLF had been hospitalized in the ICU for gastrointestinal bleeding, which was not related to portal hypertension and was not February 2014 \| Volume 9 \| Issue 2 \| e89230 PLOS ONE \| www.plosone.org 3 Metabolomic Approach of ACLF Figure 1. Flow chart of the patients. doi:10.1371/journal.pone.0089230.g001 Figure 1. Flow chart of the patients. doi:10.1371/journal.pone.0089230.g001 The model was internally validated, as all Q2Y and R2Y values obtained with the permuted Y were smaller than those from the model. Intercept values for Q2Y and R2Y, obtained from the permutation plot (not shown), were, respectively, 20.22 and 0.19. To evaluate that the difference of the group size did not influence our results, we have built 500 different models. Those OPLS models were built with 30 patients of CLF group randomly selected and 30 ACLF cases. The distribution of Q2Y values obtained described the results. The median of this Q2Y distribution was 0.64 and the smaller Q2Y value was 0.49. For each NMR variable, distribution of the regression coefficient was The model was internally validated, as all Q2Y and R2Y values obtained with the permuted Y were smaller than those from the model. OPLS Model and Their Validation Intercept values for Q2Y and R2Y, obtained from the permutation plot (not shown), were, respectively, 20.22 and 0.19. severe, one had acute kidney injury related to acute cardiac insufficiency, and one had an acute episode of seizure related to alcohol withdrawal. All these patients were alive after hospitaliza- tion in the ICU, and none had major impairment of liver function during their stay in the ICU. Two patients from the CLF group were misclassified on the score plot in the ACLF group: one had liver and kidney failure and died at 1 month after inclusion, and the other was initially classified as Child A at admission although the final diagnosis was non-severe acute alcoholic hepatitis. To evaluate that the difference of the group size did not influence our results, we have built 500 different models. Those OPLS models were built with 30 patients of CLF group randomly selected and 30 ACLF cases. The distribution of Q2Y values obtained described the results. The median of this Q2Y distribution was 0.64 and the smaller Q2Y value was 0.49. For each NMR variable, distribution of the regression coefficient was Table 1. Baseline characteristics of the overall population. Factor CLF group (n = 95) ACLF group (n = 35) Age (years)a 58.161.0 58.961.5 Male genderb 85 (84%) 27 (87%) ALT (UI/L)a 54.666.0 142.3669.5 AST (UI/L)a 94.5614.5 532.56275.5* Albumin (g/L)a 37.160.7 31.461.2* Prothrombin level (% control)a 65.762.2 34.762.8* Bilirubin (mmol/L)a 45.367.6 75.2614.4 Child–Pugh scorea 7.060.2 9.660.3* MELD scorea 13.460.6 25.661.8* Creatinine (mmol/L)a 85.363.5 158.6618.8* Blood glucose (mmol/L) 6.660.2 8.360.8* White blood-cell count (G/L)a 6.160.2 12.661.6* Paletted count (G/L)a 137.767.5 101.4612.9* Hemoglobinemia (g/L) 12.660.2 8.660.4* Note. All biological and clinical parameters were recorded at inclusion. aMean 6 SEM. bNumber (percentage) of patients. p,0.05 between ACLF and CLF groups. doi:10.1371/journal.pone.0089230.t001 Table 1. Baseline characteristics of the overall population. Table 2. Characteristics of patients with ACLF included in the OPLS analysis. Initially diagnosed in the ICU 30 GIB 12 Hemorrhagic shock 5 Severe sepsis 4 Septic shock 6 Acute kidney failure 1 Hepatic encephalopathy 2 Previous CPT 7.662.1 Admission CPT 9.762.0 Mechanical ventilation (n) 16/30 Catecholamines (n) 7/30 SOFA 7.763.6 Encephalopathy (0/I/II/III/IV) 3/11/10/1/6 Lactate (mM) 5.766.3 Death (n) 6/30 p,0.01 between previous and admission CPTs. Notes: SOFA: Sequential Organ Failure Assessment; CPT: Child–Pugh–Turcott score; GIB: gastrointestinal bleeding. doi:10.1371/journal.pone.0089230.t002 Table 2. Characteristics of patients with ACLF included in the OPLS analysis. doi:10.1371/journal.pone.0089230.t001 OPLS Model and Their Validation Initially diagnosed in the ICU 30 GIB 12 Hemorrhagic shock 5 Severe sepsis 4 Septic shock 6 Acute kidney failure 1 Hepatic encephalopathy 2 Previous CPT 7.662.1 Admission CPT 9.762.0 Mechanical ventilation (n) 16/30 Catecholamines (n) 7/30 SOFA 7.763.6 Encephalopathy (0/I/II/III/IV) 3/11/10/1/6 Lactate (mM) 5.766.3 Death (n) 6/30 p,0.01 between previous and admission CPTs. Notes: SOFA: Sequential Organ Failure Assessment; CPT: Child–Pugh–Turcott score; GIB: gastrointestinal bleeding. doi:10.1371/journal.pone.0089230.t002 Table 2. Characteristics of patients with ACLF included in the OPLS analysis. February 2014 \| Volume 9 \| Issue 2 \| e89230 PLOS ONE \| www.plosone.org 4 Metabolomic Approach of ACLF Figure 2. OPLS score plot and spectra of the CLF and ACLF patients. A: Score plot separating CLF (blue) and ACLF (red) patients. B and C: Representative spectra showing the metabolic differences between patients with CLF or ACLF. Legends: a: -CH3 of fatty acid; b; -CH2 of fatty acid; c: glucose (C) and hydroxy-ethyl starch (B); d: fumaric acid; 1: cholesterol; 2: hydroxybutyrate; 3: lactate; 4: acetoacetate; 5: pyruvate; 6: glutamine; 7: creatinine; 8 & 9: aromatic amino acids (phenylalanine and tyrosine). doi:10.1371/journal.pone.0089230.g002 Figure 2. OPLS score plot and spectra of the CLF and ACLF patients. A: Score plot separating CLF (blue) and ACLF (red) patients. B and C: Representative spectra showing the metabolic differences between patients with CLF or ACLF. Legends: a: -CH3 of fatty acid; b; -CH2 of fatty acid; c: glucose (C) and hydroxy-ethyl starch (B); d: fumaric acid; 1: cholesterol; 2: hydroxybutyrate; 3: lactate; 4: acetoacetate; 5: pyruvate; 6: glutamine; 7: creatinine; 8 & 9: aromatic amino acids (phenylalanine and tyrosine). doi:10.1371/journal.pone.0089230.g002 also estimated. The variables considered as significant in the original model were also significant in the 500 resampled models. Several amino acids could be identified in the spectra. Glutamine and glutamate were significantly higher in the ACLF group than in the CLF group. Aromatic amino acids, such tyrosine and phenylalanine, were significantly higher in the ACLF group compared to the CLF group. Creatinine was also higher in the spectra of patients in the ACLF group compared to the CLF group. Discriminants Metabolites The metabolites that differed between the groups could be identified within the sera spectra according to their loading plots (Figure 3). Table 3 presents the nine metabolites that were significantly correlated with the model and their variations according to group. February 2014 \| Volume 9 \| Issue 2 \| e89230 Discussion The buckets are labeled according to metabolite assignment, as presented in Table 3. doi:10.1371/journal.pone.0089230.g003 Figure 3. OPLS line plot showing the discriminant metabolites between patients with CLF or ACLF. Variations of metabolites are represented using a line plot between 0–9 ppm. Positive signals correspond to metabolites present at increased concentrations in the ACLF group. Conversely, negative signals correspond to metabolites present at increased concentrations in CLF group. The buckets are labeled according to metabolite assignment, as presented in Table 3. doi:10.1371/journal.pone.0089230.g003 good hepatic function. The liver plays a key role in lipid metabolism, and low HDL levels seem to be caused by the severity of the chronic liver disease rather than indicating an acute episode of chronic impairment. A rise of serum-lactate concen- tration is common in cases of acute liver failure and has been proposed as a prognostic marker [24]. The production of lactate is also impaired in chronic liver failure accompanied by a decrease in hepatic pyruvate dehydrogenase activity [25]. Moreover, lactate is a major biomarker for tissue hypoxia and necrosis. Hepatic necrosis and inflammation, detected by histological examination of a liver biopsy, correlate well with the severity of ACLF and a poor outcome [26]. In our previous study, lactate was a key-indicator metabolite in patients with cirrhosis and the highest Model for End-Stage Liver-Disease scores. In patients with ACLF, increased lactate concentration may be related to the superimposed metabolomic profile than determine in CLF group and in our previous study (6). In the ACLF group, lipid metabolism, lactate, amino-acid metabolism, and urea metabolism were affected by impaired liver function. This point is important as it differentiated the metabolomic fingerprints of chronic liver failure in acute-on- chronic liver failure. In the CLF group, HDL signal were higher than in the ACLF group, but there was no difference with regards to VLDL and LDL. This is probably because ACLF patients were not fasted whereas those in the CLF group were. Thus, this may have caused the lipid signals to be higher in ACLF sera. Despite this discrepancy, the resonances corresponding to lipids were not increased in ACLF patients. Moreover, the HDL-lipid region was lower in ACLF sera compared to that of fasting patients in the CLF group. In several studies, higher HDL levels seem to reflect Table 3. Discriminant metabolites observed by 1H-NMR spectroscopy. Table 3. Discriminant metabolites observed by 1H-NMR spectroscopy. Table 3. Discussion In the 1H-NMR spectra of sera, the lipids were detected as broad resonances of fatty-acid methyl and methylene moieties at 0.8 and 1.24 ppm, respectively, and the N-trimethyl moiety of choline was included in phospholipids at 3.2 ppm. The line shape of the methyl and methylene fatty-acid resonances, at 0.8 and 1.24 ppm, depended on the size of the lipoprotein particles. It has been widely reported that high-density lipid (HDL) particles produce resonances with lower chemical shifts than low-density lipoprotein (LDL and VLDL) particles. Interestingly, lipoproteins with higher densities were significantly elevated in the CLF group. For VLDL, corresponding to the highest chemical shifts in methyl and methylene resonances, no differences were found. This preliminary study was conducted in a cohort of cirrhotic patients hospitalized in ICU for acute event and cirrhotic outpatients with compensated or decompensated but stable liver function. This investigation clearly shows a difference in the metabolomic profiles of the two studied populations. The comparison of these two populations of cirrhotic patients was preferred than comparison of ACLF cirrhotic patient and healthy volunteers. In the last case, metabolomics difference should be only due to chronic liver failure and not to acute impairment of the liver function. During the evolution of chronic liver failure, several changes in liver metabolism were detected. In our previous study, stable patients with alcoholic cirrhosis and various degrees of liver injury (evaluated by the MELD score) were included. In severe chronic liver failure, metabolism of lipids, glucose, and ketone bodies were significantly discriminants [6]. Interestingly, in the present study, the metabolomic profile of ACLF patients was not the same Lactate was an important metabolite that discriminated between the ACLF and CLF groups. Patients with ACLF had significantly higher lactate signals than patients in the CLF group. Pyruvate and ketone bodies (mainly acetoacetate) were signifi- cantly higher in the ACLF group than in CLF group. PLOS ONE \| www.plosone.org February 2014 \| Volume 9 \| Issue 2 \| e89230 5 February 2014 \| Volume 9 \| Issue 2 \| e89230 Metabolomic Approach of ACLF Figure 3. OPLS line plot showing the discriminant metabolites between patients with CLF or ACLF. Variations of metabolites are represented using a line plot between 0–9 ppm. Positive signals correspond to metabolites present at increased concentrations in the ACLF group. Conversely, negative signals correspond to metabolites present at increased concentrations in CLF group. singlet; b, broad; d, doublet; dd, doublet of doublet; t, triplet, m multiplet; q, quadriplet. Discussion Discriminant metabolites observed by 1H-NMR spectroscopy. No. of metabolite Name Chemical shift (ppm) and multiplicity$ r# p Group 1 Lipids (mainly HDL) 0.8b; 1.2b 20.46 4,861028 CLF 2 Hydroxybutyrate 1.13d +0.38 1.761026 ACLF 3 Lactate 1.30d; 4.13q +0.55 2.6610211 ACLF 4 Acetoacetate 2.11s +0.43 3.161027 ACLF 5 Pyruvate 2.36s +0.54 4.9610211 ACLF 6 Glutamine, glutamate 2.42m +0.48 9.661029 ACLF 7 Creatinine 3.03s 4.04s +0.56 1.1610212 ACLF 8 Tyrosine 6.88m;7.18d +0.45 9.061028 ACLF 9 Phenylalanine 7.31m; 7.40m +0.63 3.3610215 ACLF The principal discriminant metabolites are ordered according to the loading plots of patients with CLF and ACLF. The number of the metabolite corresponds to the number in the line plot in Figure 2. The chemical shift and multiplicity correspond to those found in the 1H-NMR spectra of the patients’ sera. $ppm: parts per million. s, singlet; b, broad; d, doublet; dd, doublet of doublet; t, triplet, m multiplet; q, quadriplet. #A positive correlation (r) indicates an increased concentration in the ACLF group and a negative correlation indicates a decreased concentration in the CLF group. A p-value ,2.461026 is significant. doi:10.1371/journal.pone.0089230.t003 The principal discriminant metabolites are ordered according to the loading plots of patients with CLF and ACLF. The number of the metabolite corresponds to the number in the line plot in Figure 2. The chemical shift and multiplicity correspond to those found in the 1H-NMR spectra of the patients’ sera. $ppm: parts per million. s, singlet; b, broad; d, doublet; dd, doublet of doublet; t, triplet, m multiplet; q, quadriplet. #A positive correlation (r) indicates an increased concentration in the ACLF group and a negative correlation indicates a decreased concentration in the CLF group. A p-value ,2.461026 is significant. doi:10.1371/journal.pone.0089230.t003 February 2014 \| Volume 9 \| Issue 2 \| e89230 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 6 Metabolomic Approach of ACLF impairment of hepatic lactate catabolism and increased produc- tion of lactate caused by hypoxia and liver necrosis. Ketone bodies (including hydroxybutyrate and acetoacetate) were also increased in ACLF sera. These metabolites mainly originate from the metabolism of fatty acids via the beta-oxidation pathway, which produces acetyl moieties that enter the TCA cycle. Taken together, a decrease in HDL and an increase in lactate and ketone bodies have led to the hypothesis that aerobic glycolysis is modulated towards anaerobic and lipid metabolisms. Discussion The main limitation encountered in a translational metabolo- mic approach to liver disease is methodological bias when selecting patients, which limits the confidence that can be drawn from conclusions. We attempted to avoid these pitfalls by constructing well-defined subgroups of patients according to their stage of underlying liver disease, with a control group composed of compensated or uncompensated (CLF group), but stable, cirrhosis, and a group of patients with cirrhosis who had an acute event that was responsible for the superimposed liver impairment (ACLF group). We also focused specifically on the etiology of alcoholism to avoid any potential specific influence of viral or non-alcoholic- related liver disease on the metabolomic profiles of the sera, as has been previously reported [10]. The perturbation of the aromatic acids metabolism (phenylal- anine and tyrosine metabolism), has been found in different etiologies of liver injury, such as traumatic injury, and seems to be early markers [27]. In patients with chronic liver disease and who have had an acute event, such as gastrointestinal bleeding, infection, acute alcoholic hepatitis, or encephalopathy, the aromatic acids are increased in relation to inflammation and hepatic necrosis [28]. Systemic inflammation occurs in patients with cirrhosis who have a superimposed liver insult, and is significantly related to bleeding, sepsis, and long-term outcomes [29]. Inflammation plays a key role in worsening the patient’s health, and elevated levels of multiple pro-inflammatory cytokines have been described in ACLF [30]. In this setting, the present data enabled us to form several hypotheses and conclusions. First, the metabolomic fingerprints reflect major changes to liver function, such as energy metabolism, amino-acid metabolism, and urea metabolism. Secondly, in cases of acute impairment, the changes correlated with the severity of liver failure (as shown in our previous study) and they were increased. Other modulations were not found in chronic liver failure and increase in case of acute dysfunction of the liver such as the aromatic acids. Third, increase creatinine in the ACLF group reflects extra-liver failure with renal injury comparatively of cirrhotic patients of the CLF group. Fourth, the metabolomic changes were related to inflammation and necrosis. Systemic inflammation and necrosis are known to play a major role in the pathophysiology of acute-on-chronic liver failure and the failure of other organs. In addition, our metabolomic profiles improved the classification of patients with or without acute-on-chronic liver failure, as was shown with the initially misclassified patients. 6. Amathieu R, Nahon P, Triba M, Bouchemal N, Trinchet JC, et al. (2011) Metabolomic approach by 1H NMR spectroscopy of serum for the assessment of chronic liver failure in patients with cirrhosis. Journal of proteome research 10: 3239–3245. 9. Jimenez B, Montoliu C, MacIntyre DA, Serra MA, Wassel A, et al. (2010) Serum metabolic signature of minimal hepatic encephalopathy by (1)H-nuclear magnetic resonance. Journal of proteome research 9: 5180–5187. 8. Yu K, Sheng G, Sheng J, Chen Y, Xu W, et al. (2007) A metabonomic investigation on the biochemical perturbation in liver failure patients caused by hepatitis B virus. Journal of proteome research 6: 2413–2419. 7. Martinez-Granados B, Morales JM, Rodrigo JM, Del Olmo J, Serra MA, et al. (2011) Metabolic profile of chronic liver disease by NMR spectroscopy of human biopsies. International journal of molecular medicine 27: 111–117. Discussion The liver is the major organ for urea metabolism. Impaired urea metabolism leads to decreased ammonia detoxification and is responsible for the increase levels of glutamine and glutamate. This increase occurs in different situations in patients with chronic liver diseases: gastrointestinal bleeding, infection [28], hepatic encephalopathy, or end-stage liver disease [6]. Then, our metabolomic approach identifies several indicative factors within one experiment, whereas these factors have been only previously described in a range of situations in patients with cirrhosis. The fact that lactate and creatinine, for example, represents established biomarkers in the management of decom- pensated cirrhosis is not novel at all. The improvement reach by the metabolomics approach could be the simultaneous evaluation of the metabolites of interest in a profile directly correlated to the clinical status. A metabolomic approach assesses the liver markers and other organ impairments in serum samples using a unique experiment: this profiling provides complete and multivariate information on the metabolomic changes related to ACLF and can stratify the cases. The use of this technique as a diagnostic and prognostic tool for patients with ACLF needs to be further investigated. Finally, further confirmation of our metabolomic findings needs to be tested in larger prospective cohorts of ICU patients being followed up for cirrhosis in order to better define clear association between metabolomic profiles and diagnosis or prognosis. Such an approach will improve our understanding of the involvement of the biological pathways in ACLF. Two patients within our CLF group were misclassified in the score plot and had similar metabolomic profile than patients of ACLF group. Both patients had impaired liver function: this was caused by acute alcoholic hepatitis for one, and cardiac failure for the other. Metabolomic fingerprints seemed able to reflect changes in liver function before standard biological laboratory tests could. In addition, four patients from the ACLF group were misclassified as being in the CLF group. They had minor gastrointestinal bleeding, acute kidney injury, encephalopathy that was not related to liver disease, and none or minor liver injury. Metabolomic profiles could excluded acute liver damage better than standard biological laboratory test. 10. Qi S, Tu Z, Ouyang X, Wang L, Peng W, et al. (2012) Comparison of the metabolic profiling of hepatitis B virus-infected cirrhosis and alcoholic cirrhosis Author Contributions Conceived and designed the experiments: RA MNT PN LLM. Performed the experiments: RA MNT HH WK NB LLM. Analyzed the data: RA MNT LLM NB. Contributed reagents/materials/analysis tools: RA MNT LLM NB PS GD JCT. Wrote the paper: RA MNT LLM PN GD JCT. Conceived and designed the experiments: RA MNT PN LLM. Performed the experiments: RA MNT HH WK NB LLM. Analyzed the data: RA MNT LLM NB. Contributed reagents/materials/analysis tools: RA MNT LLM NB PS GD JCT. Wrote the paper: RA MNT LLM PN GD JCT. 2. Jalan R, Williams R (2002) Acute-on-chronic liver failure: pathophysiological basis of therapeutic options. Blood purification 20: 252–261. 1. Olson JC, Wendon JA, Kramer DJ, Arroyo V, Jalan R, et al. (2011) Intensive care of the patient with cirrhosis. Hepatology 54: 1864–1872. Metabolomic Approach of ACLF patients by using (1) H NMR-based metabonomics. 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NMR in biomedicine 19: 521–526. 25. Shangraw RE, Rabkin JM, Lopaschuk GD (1998) Hepatic pyruvate dehydro- genase activity in humans: effect of cirrhosis, transplantation, and dichloroace- tate. The American journal of physiology 274: G569–577. 15. Dellinger RP, Levy MM, Carlet JM, Bion J, Parker MM, et al. (2008) Surviving Sepsis Campaign: international guidelines for management of severe sepsis and septic shock: 2008. Critical care medicine 36: 296–327. 26. Rastogi A, Kumar A, Sakhuja P, Bihari C, Gondal R, et al. References 1. Olson JC, Wendon JA, Kramer DJ, Arroyo V, Jalan R, et al. (2011) Intensive care of the patient with cirrhosis. Hepatology 54: 1864–1872. 2. Jalan R, Williams R (2002) Acute-on-chronic liver failure: pathophysiological basis of therapeutic options. Blood purification 20: 252–261. 3. Moreau R, Jalan R, Gines P, Pavesi M, Angeli P, et al. (2013) Acute-on-chronic liver failure is a distinct syndrome that develops in patients with acute decompensation of cirrhosis. Gastroenterology 144: 1426–1437, 1437 e1421– 1429. 4. Sarin SK, Kumar A, Almeida JA, Chawla YK, Fan ST, et al. (2009) Acute-on- chronic liver failure: consensus recommendations of the Asian Pacific Association for the study of the liver (APASL). Hepatology international 3: 269–282. 5. Dunn WB, Broadhurst DI, Atherton HJ, Goodacre R, Griffin JL (2011) Systems level studies of mammalian metabolomes: the roles of mass spectrometry and nuclear magnetic resonance spectroscopy. Chemical Society reviews 40: 387– 426. 5. Dunn WB, Broadhurst DI, Atherton HJ, Goodacre R, Griffin JL (2011) Systems level studies of mammalian metabolomes: the roles of mass spectrometry and nuclear magnetic resonance spectroscopy. Chemical Society reviews 40: 387– 426. February 2014 \| Volume 9 \| Issue 2 \| e89230 7 PLOS ONE \| www.plosone.org Metabolomic Approach of ACLF Metabolomic Approach of ACLF (2011) Liver histology as predictor of outcome in patients with acute-on-chronic liver failure (ACLF). Virchows Archiv : an international journal of pathology 459: 121–127. 16. Vincent JL, de Mendonca A, Cantraine F, Moreno R, Takala J, et al. (1998) Use of the SOFA score to assess the incidence of organ dysfunction/failure in intensive care units: results of a multicenter, prospective study. Working group on ‘‘sepsis-related problems’’ of the European Society of Intensive Care Medicine. Critical care medicine 26: 1793–1800. 27. Ranjan P, Gupta A, Kumar S, Gowda GA, Ranjan A, et al. (2006) Detection of new amino acid markers of liver trauma by proton nuclear magnetic resonance spectroscopy. Liver international : official journal of the International Association for the Study of the Liver 26: 703–707. y 28. Rosen HM, Yoshimura N, Hodgman JM, Fischer JE (1977) Plasma amino acid patterns in hepatic encephalopathy of differing etiology. Gastroenterology 72: 483–487. 17. 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Liver transplantation : official publication of the American Association for the Study of Liver Diseases and the International Liver Transplantation Society 10: 1109–1119. 20. Nicholson JK, Foxall PJ, Spraul M, Farrant RD, Lindon JC (1995) 750 MHz 1H and 1H-13C NMR spectroscopy of human blood plasma. Anal Chem 67: 793–811. February 2014 \| Volume 9 \| Issue 2 \| e89230 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 8 8
https://openalex.org/W2968178196	https://jmir.org/api/download?alt_name=mental_v7i1e13392_app3.pdf&filename=52fdc02b804f311f6663a167ea8f3763.pdf	English	null	A Culturally Adapted Cognitive Behavioral Internet-Delivered Intervention for Depressive Symptoms: Randomized Controlled Trial	JMIR mental health	2,020	cc-by	16,882	7/17/2019 7/17/2019 CONSORT-EHEALTH (V 1.6.1) - Submission/Publication Form CONSORT-EHEALTH (V 1.6.1) - Submission/Publication Form In the textboxes, either copy & paste the relevant sections from your manuscript into this form - please include any quotes from your manuscript in QUOTATION MARKS, please include any quotes from your manuscript in QUOTATION MARKS, or answer directly by providing additional information not in the manuscript, or elaborating on why the item was not relevant for this study. or answer directly by providing additional information not in the manuscript, or elaborating on why the item was not relevant for this study. YOUR ANSWERS WILL BE PUBLISHED AS A SUPPLEMENTARY FILE TO YOUR PUBLICATION IN JMIR AND ARE CONSIDERED PART OF YOUR PUBLICATION (IF ACCEPTED). Please ¦ll in these questions diligently. Information will not be copyedited, so please use proper spelling and grammar, use correct capitalization, and avoid abbreviations. DO NOT FORGET TO SAVE AS PDF _AND_ CLICK THE SUBMIT BUTTON SO YOUR ANSWERS ARE IN OUR DATABASE !!! CONSORT-EHEALTH (V 1.6.1) - Submission/Publication Form The CONSORT-EHEALTH checklist is intended for authors of randomized trials evaluating web- based and Internet-based applications/interventions, including mobile interventions, electronic games (incl multiplayer games), social media, certain telehealth applications, and other interactive and/or networked electronic applications. Some of the items (e.g. all subitems under item 5 - description of the intervention) may also be applicable for other study designs. The goal of the CONSORT EHEALTH checklist and guideline is to be a) a guide for reporting for authors of RCTs, b) to form a basis for appraisal of an ehealth trial (in terms of validity) CONSORT-EHEALTH items/subitems are MANDATORY reporting items for studies published in the Journal of Medical Internet Research and other journals / scienti¦c societies endorsing the checklist. Items numbered 1., 2., 3., 4a., 4b etc are original CONSORT or CONSORT-NPT (non-pharmacologic treatment) items. Items with Roman numerals (i., ii, iii, iv etc.) are CONSORT-EHEALTH extensions/clari¦cations. As the CONSORT-EHEALTH checklist is still considered in a formative stage, we would ask that you also RATE ON A SCALE OF 1-5 how important/useful you feel each item is FOR THE PURPOSE OF THE CHECKLIST and reporting guideline (optional). As the CONSORT-EHEALTH checklist is still considered in a formative stage, we would ask that you also RATE ON A SCALE OF 1-5 how important/useful you feel each item is FOR THE PURPOSE OF THE CHECKLIST and reporting guideline (optional). Mandatory reporting items are marked with a red . In the textboxes, either copy & paste the relevant sections from your manuscript into this form - please include any quotes from your manuscript in QUOTATION MARKS, or answer directly by providing additional information not in the manuscript, or elaborating on why the item was not relevant for this study. Mandatory reporting items are marked with a red . In the textboxes, either copy & paste the relevant sections from your manuscript into this form - please include any quotes from your manuscript in QUOTATION MARKS, or answer directly by providing additional information not in the manuscript, or elaborating on why the item was not relevant for this study. Mandatory reporting items are marked with a red . DO NOT FORGET TO SAVE AS PDF _AND_ CLICK THE SUBMIT BUTTON SO YOUR ANSWERS ARE IN OUR DATABASE !!! Citation Suggestion (if you append the pdf as Appendix we suggest to cite this paper in the caption): CONSORT-EHEALTH: Improving and Standardizing Evaluation Reports of Web-based and Mobile Health Interventions J Med Internet Res 2011;13(4):e126 URL: http://www.jmir.org/2011/4/e126/ doi: 10.2196/jmir.1923 PMID 22209829 CONSORT-EHEALTH: Improving and Standardizing Evaluation Reports of Web-based and Mobi Health Interventions J Med Internet Res 2011;13(4):e126 URL: http://www.jmir.org/2011/4/e126/ doi: 10.2196/jmir.1923 PMID: 22209829 Required rms/d/e/1FAIpQLSfZBSUp1bwOc_OimqcS64RdfIAFvmrTSkZQL2-3O8O9hrL5Sw/viewform?hl=en_US&formkey=dGl… 1/47 https://docs.google.com/forms/d/e/1FAIpQLSfZBSUp1bwOc_OimqcS64RdfIAFvmrTSkZQL2-3O8O9hrL5Sw/viewform? 9 CONSORT-EHEALTH (V 1.6.1) - Submission/Publication Form Your name First Last Alicia Salamanca-Sanabria Primary A¨liation (short), City, Country * University of Toronto, Toronto, Canada Trinity College Dublin Your e-mail address * abc@gmail.com salamana@tcd.ie Title of your manuscript * Provide the (draft) title of your manuscript. A Randomised Control Trial of a Culturally Adapted Cognitive Behavioural Internet-delivered treatment for College Students with Depressive Symptoms Name of your App/Software/Intervention * If there is a short and a long/alternate name, write the short name ¦rst and add the long name in brackets. Space from depression /SilverCloud Hea Evaluated Version (if any) e.g. "V1", "Release 2017-03-01", "Version 2.0.27913" V1,"2019-04-24", V2, "2019-04-26" 7/17/2019 CONSORT-EHEALTH (V 1.6.1) - Submission/Publication Form A Randomised Control Trial of a Culturally Adapted Cognitive Behavioural Internet-delivered treatment for College Students with Depressive Symptoms 2/47 CONSORT-EHEALTH (V 1.6.1) - Submission/Publication Form access is free and open access only for special usergroups, not open access is open to everyone, but requires payment/subscription/in-app purchases app/intervention no longer accessible Other: Language(s) * What language is the intervention/app in? If multiple languages are available, separate by comma (e.g. "English, French") English, Spanish URL of your Intervention Website or App e.g. a direct link to the mobile app on app in appstore (itunes, Google Play), or URL of the website. If the intervention is a DVD or hardware, you can also link to an Amazon page. https://es.silvercloudhealth.com/ URL of an image/screenshot (optional) Your answer Accessibility * Can an enduser access the intervention presently? Primary Medical Indication/Disease/Condition * e.g. "Stress", "Diabetes", or de¦ne the target group in brackets after the condition, e.g. "Autism (Parents of children with)", "Alzheimers (Informal Caregivers of)" Depression URL of an image/screenshot (optional) Your answer CONSORT-EHEALTH (V 1.6.1) - Submission/Publication Form access is free and open access only for special usergroups, not open access is open to everyone, but requires payment/subscription/in-app purchases app/intervention no longer accessible Other: Language(s) * What language is the intervention/app in? If multiple languages are available, separate by comma (e.g. "English, French") English, Spanish URL of your Intervention Website or App e.g. a direct link to the mobile app on app in appstore (itunes, Google Play), or URL of the website. If the intervention is a DVD or hardware, you can also link to an Amazon page. https://es.silvercloudhealth.com/ URL of an image/screenshot (optional) Your answer Accessibility * Can an enduser access the intervention presently? Primary Medical Indication/Disease/Condition * e.g. "Stress", "Diabetes", or de¦ne the target group in brackets after the condition, e.g. "Autism (Parents of children with)", "Alzheimers (Informal Caregivers of)" Depression CONSORT-EHEALTH (V 1.6.1) - Submission/Publication Form 7/17/2019 English, Spanish Accessibility * 3/47 rms/d/e/1FAIpQLSfZBSUp1bwOc_OimqcS64RdfIAFvmrTSkZQL2-3O8O9hrL5Sw/viewform?hl=en_US&formkey=dGl… https://docs.google.com/forms/d/e/1FAIpQLSfZBSUp1bwOc_OimqcS64RdfIAFvmrTSkZQL2-3O8O9hrL5Sw/viewform? 7/17/2019 CONSORT-EHEALTH (V 1.6.1) - Submission/Publication Form CONSORT EHEALTH (V 1.6.1) Submission/Publication Form Approximately Daily Approximately Weekly Approximately Monthly Approximately Yearly "as needed" Other: Primary Outcomes measured in trial * comma-separated list of primary outcomes reported in the trial Depressive symptoms Secondary/other outcomes Are there any other outcomes the intervention is expected to affect? Anxiety symptoms Recommended "Dose" * What do the instructions for users say on how often the app should be used? Primary Outcomes measured in trial * comma-separated list of primary outcomes reported in the trial Depressive symptoms Primary Outcomes measured in trial * comma-separated list of primary outcomes reported in the trial Anxiety symptoms Recommended "Dose" * What do the instructions for users say on how often the app should be used? Other: rms/d/e/1FAIpQLSfZBSUp1bwOc_OimqcS64RdfIAFvmrTSkZQL2-3O8O9hrL5Sw/viewform?hl=en_US&formkey=dGl… https://docs.google.com/forms/d/e/1FAIpQLSfZBSUp1bwOc_OimqcS64RdfIAFvmrTSkZQL2-3O8O9hrL5Sw/viewform? 4/47 7/17/2019 CONSORT-EHEALTH (V 1.6.1) - Submission/Publication Form unknown / not evaluated 0-10% 11-20% 21-30% 31-40% 41-50% 51-60% 61-70% 71%-80% 81-90% 91-100% Other: Approx. Percentage of Users (starters) still using the app as recommended after 3 months * Approx. Percentage of Users (starters) still using the app as recommended after 3 months * 91-100% rms/d/e/1FAIpQLSfZBSUp1bwOc_OimqcS64RdfIAFvmrTSkZQL2-3O8O9hrL5Sw/viewform?hl=en_US&formkey=dGl… https://docs.google.com/forms/d/e/1FAIpQLSfZBSUp1bwOc_OimqcS64RdfIAFvmrTSkZQL2-3O8O9hrL5Sw/viewform? 5/47 CONSORT-EHEALTH (V 1.6.1) - Submission/Publication Form yes: all primary outcomes were signi¦cantly better in intervention group vs control partly: SOME primary outcomes were signi¦cantly better in intervention group vs control no statistically signi¦cant difference between control and intervention potentially harmful: control was signi¦cantly better than intervention in one or more outcomes inconclusive: more research is needed Other: not submitted yet - in early draft status not submitted yet - in late draft status, just before submission submitted to a journal but not reviewed yet submitted to a journal and after receiving initial reviewer comments submitted to a journal and accepted, but not published yet published Other: Overall, was the app/intervention effective? * Article Preparation Status/Stage * At which stage in your article preparation are you currently (at the time you ¦ll in this form) CONSORT-EHEALTH (V 1.6.1) - Submission/Publication Form 7/17/2019 yes: all primary outcomes were signi¦cantly better in intervention group vs control partly: SOME primary outcomes were signi¦cantly better in intervention group vs control no statistically signi¦cant difference between control and intervention potentially harmful: control was signi¦cantly better than intervention in one or more outcomes inconclusive: more research is needed Other: not submitted yet - in early draft status not submitted yet - in late draft status, just before submission submitted to a journal but not reviewed yet submitted to a journal and after receiving initial reviewer comments submitted to a journal and accepted, but not published yet published Other: Overall, was the app/intervention effective? * Article Preparation Status/Stage * At which stage in your article preparation are you currently (at the time you ¦ll in this form) yes: all primary outcomes were signi¦cantly better in intervention group vs control partly: SOME primary outcomes were signi¦cantly better in intervention group vs control no statistically signi¦cant difference between control and intervention potentially harmful: control was signi¦cantly better than intervention in one or more outcomes inconclusive: more research is needed Other: Overall, was the app/intervention effective? * Overall, was the app/intervention effective? * rms/d/e/1FAIpQLSfZBSUp1bwOc_OimqcS64RdfIAFvmrTSkZQL2-3O8O9hrL5Sw/viewform?hl=en_US&formkey=dGl… https://docs.google.com/forms/d/e/1FAIpQLSfZBSUp1bwOc_OimqcS64RdfIAFvmrTSkZQL2-3O8O9hrL5Sw/viewform? 6/47 CONSORT-EHEALTH (V 1.6.1) - Submission/Publication Form not submitted yet / unclear where I will submit this Journal of Medical Internet Research (JMIR) JMIR mHealth and UHealth JMIR Serious Games JMIR Mental Health JMIR Public Health JMIR Formative Research Other JMIR sister journal Other: Pilot/feasibility Fully powered no ms number (yet) / not (yet) submitted to / published in JMIR Other: Journal * If you already know where you will submit this paper (or if it is already submitted), please provide the journal name (if it is not JMIR, provide the journal name under "other") Is this a full powered effectiveness trial or a pilot/feasibility trial? * Manuscript tracking number * If this is a JMIR submission, please provide the manuscript tracking number under "other" (The ms tracking number can be found in the submission acknowledgement email, or when you login as author in JMIR. If the paper is already published in JMIR, then the ms tracking number is the four- digit number at the end of the DOI, to be found at the bottom of each published article in JMIR) CONSORT-EHEALTH (V 1.6.1) - Submission/Publication Form not submitted yet / unclear where I will submit this Journal of Medical Internet Research (JMIR) JMIR mHealth and UHealth JMIR Serious Games JMIR Mental Health JMIR Public Health JMIR Formative Research Other JMIR sister journal Other: Pilot/feasibility Fully powered no ms number (yet) / not (yet) submitted to / published in JMIR Other: TITLE AND ABSTRACT Journal * If you already know where you will submit this paper (or if it is already submitted), please provide the journal name (if it is not JMIR, provide the journal name under "other") Is this a full powered effectiveness trial or a pilot/feasibility trial? * Manuscript tracking number * If this is a JMIR submission, please provide the manuscript tracking number under "other" (The ms tracking number can be found in the submission acknowledgement email, or when you login as author in JMIR. If the paper is already published in JMIR, then the ms tracking number is the four- digit number at the end of the DOI, to be found at the bottom of each published article in JMIR) 7/17/2019 CONSORT-EHEALTH (V 1.6.1) - Submission/Publication Form Journal * If you already know where you will submit this paper (or if it is already submitted), please provide the journal name (if it is not JMIR, provide the journal name under "other") not submitted yet / unclear where I will submit this Manuscript tracking number * g If this is a JMIR submission, please provide the manuscript tracking number under "other" (The ms tracking number can be found in the submission acknowledgement email, or when you login as author in JMIR. If the paper is already published in JMIR, then the ms tracking number is the four- digit number at the end of the DOI, to be found at the bottom of each published article in JMIR) no ms number (yet) / not (yet) submitted to / published in JMIR Other: TITLE AND ABSTRACT TITLE AND ABSTRACT https://docs.google.com/forms/d/e/1FAIpQLSfZBSUp1bwOc_OimqcS64RdfIAFvmrTSkZQL2-3O8O9hrL5Sw/viewform?hl=en_US&formkey=dGl… 7/47 https://docs.google.com/forms/d/e/1FAIpQLSfZBSUp1bwOc_OimqcS64RdfIAFvmrTSkZQL2-3O8O9hrL5Sw/viewform? CONSORT-EHEALTH (V 1.6.1) - Submission/Publication Form 7/17/2019 1a) TITLE: Identi¦cation as a randomized trial in the title 1a) yes Other: 1a) Does your paper address CONSORT item 1a? * I.e does the title contain the phrase "Randomized Controlled Trial"? (if not, explain the reason under "other") yes Other: 1a) Does your paper address CONSORT item 1a? * I.e does the title contain the phrase "Randomized Controlled Trial"? (if not, explain the reason under "other") 1a-i) Identify the mode of delivery in the title Identify the mode of delivery. Preferably use “web-based” and/or “mobile” and/or “electronic game” in the title. Avoid ambiguous terms like “online”, “virtual”, “interactive”. Use “Internet-based” only if Intervention includes non-web-based Internet components (e.g. email), use “computer-based” or “electronic” only if o©ine products are used. Use “virtual” only in the context of “virtual reality” (3-D worlds). Use “online” only in the context of “online support groups”. Complement or substitute product names with broader terms for the class of products (such as “mobile” or “smart phone” instead of “iphone”), especially if the application runs on different platforms. subitem not at all important 1 2 3 4 5 essential subitem not at all important Does your paper address subitem 1a-i? * 7/17/2019 CONSORT-EHEALTH (V 1.6.1) - Submission/Publication Form subitem not at all important 1 2 3 4 5 essential 1b) ABSTRACT: Structured summary of trial design, methods, results, and conclusions NPT extension: Description of experimental treatment, comparator, care providers, centers, and blinding status Does your paper address subitem 1a-ii? Copy and paste relevant sections from manuscript title (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or brie§y explain why the item is not applicable/relevant for your study Your answer 1a-iii) Primary condition or target group in the title Mention primary condition or target group in the title, if any (e.g., “for children with Type I Diabetes”) Example: A Web-based and Mobile Intervention with Telephone Support for Children with Type I Diabetes: Randomized Controlled Trial Does your paper address subitem 1a-iii? * Copy and paste relevant sections from manuscript title (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or brie§y explain why the item is not applicable/relevant for your study A Randomised Control Trial of a Culturally Adapted Cognitive Behavioural Internet-delivered treatment for Depressive Symptoms 7/17/2019 CONSORT-EHEALTH (V 1.6.1) - Submission/Publication Form Does your paper address subitem 1a-ii? y Copy and paste relevant sections from manuscript title (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or brie§y explain why the item is not applicable/relevant for your study Your answer subitem not at all 1 2 3 4 5 essential 1a-iii) Primary condition or target group in the title Mention primary condition or target group in the title, if any (e.g., “for children with Type I Diabetes”) Example: A Web-based and Mobile Intervention with Telephone Support for Children with Type I Diabetes: Randomized Controlled Trial Does your paper address subitem 1a-i? * Copy and paste relevant sections from manuscript title (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or brie§y explain why the item is not applicable/relevant for your study A Randomised Control Trial of a Culturally Adapted Cognitive Behavioural Internet-delivered treatment for Depressive Symptoms 8/47 Does your paper address subitem 1a-iii? * y p p Copy and paste relevant sections from manuscript title (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or brie§y explain why the item is not applicable/relevant for your study A Randomised Control Trial of a Culturally Adapted Cognitive Behavioural Internet-delivered treatment for Depressive Symptoms 1b) ABSTRACT: Structured summary of trial design, methods, results, and conclusions NPT extension: Description of experimental treatment, comparator, care providers, centers, and blinding status. 9/47 rms/d/e/1FAIpQLSfZBSUp1bwOc_OimqcS64RdfIAFvmrTSkZQL2-3O8O9hrL5Sw/viewform?hl=en_US&formkey=dGl… https://docs.google.com/forms/d/e/1FAIpQLSfZBSUp1bwOc_OimqcS64RdfIAFvmrTSkZQL2-3O8O9hrL5Sw/viewform? 7/17/2019 CONSORT-EHEALTH (V 1.6.1) - Submission/Publication Form ABSTRACT Mention key features/functionalities/components of the intervention and comparator in the abstract. If possible, also mention theories and principles used for designing the site. Keep in mind the needs of systematic reviewers and indexers by including important synonyms. (Note: Only report in the abstract what the main paper is reporting. If this information is missing from the main body of text, consider adding it) subitem not at all important 1 2 3 4 5 essential 1b-ii) Level of human involvement in the METHODS section of the ABSTRACT Clarify the level of human involvement in the abstract, e.g., use phrases like “fully automated” vs. “therapist/nurse/care provider/physician-assisted” (mention number and expertise of providers involved, if any). (Note: Only report in the abstract what the main paper is reporting. If this information is missing from the main body of text, consider adding it) subitem not at all important 1 2 3 4 5 essential nformation is missing from the main body of text, consider adding it) Does your paper address subitem 1b-i? * Copy and paste relevant sections from the manuscript abstract (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or brie§y explain why the item is not applicable/relevant for your study "cognitive behavioural internet-delivered treatment" "The programme consisted of 7-modules". "Participants received weekly support from a trained supporter" Does your paper address subitem 1b-ii? Copy and paste relevant sections from the manuscript abstract (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or brie§y explain why the item is not applicable/relevant for your study Participants received weekly support from a trained supporter https://docs.google.com/forms/d/e/1FAIpQLSfZBSUp1bwOc_OimqcS64RdfIAFvmrTSkZQL2-3O8O9hrL5Sw/viewform?hl=en_US&formkey=dG… 10/47 https://docs.google.com/forms/d/e/1FAIpQLSfZBSUp1bwOc_OimqcS64RdfIAFvmrTSkZQL2-3O8O9hrL5Sw/viewform? m/forms/d/e/1FAIpQLSfZBSUp1bwOc_OimqcS64RdfIAFvmrTSkZQL2-3O8O9hrL5Sw/viewform?hl=en_US&formkey=dG 7/17/2019 CONSORT-EHEALTH (V 1.6.1) - Submission/Publication Form 1b-iii) Open vs. closed, web-based (self-assessment) vs. face-to- face assessments in the METHODS section of the ABSTRACT 1b-iii) Open vs. closed, web-based (self-assessment) vs. face-to- face assessments in the METHODS section of the ABSTRACT Mention how participants were recruited (online vs. o©ine), e.g., from an open access website or from a clinic or a closed online user group (closed usergroup trial), and clarify if this was a purely web-based trial, or there were face-to-face components (as part of the intervention or for assessment). Clearly say if outcomes were self-assessed through questionnaires (as common in web-based trials). Note: In traditional o©ine trials, an open trial (open-label trial) is a type of clinical trial in which both the researchers and participants know which treatment is being administered. To avoid confusion, use “blinded” or “unblinded” to indicated the level of blinding instead of “open”, as “open” in web-based trials usually refers to “open access” (i.e. participants can self-enrol). (Note: Only report in the abstract what the main paper is reporting. If this information is missing from the main body of text, consider adding it) subitem not at all important 1 2 3 4 5 essential 1b-iv) RESULTS section in abstract must contain use data 1b-iv) RESULTS section in abstract must contain use data Report number of participants enrolled/assessed in each group, the use/uptake of the intervention (e.g., attrition/adherence metrics, use over time, number of logins etc.), in addition to primary/secondary outcomes. (Note: Only report in the abstract what the main paper is reporting. If this information is missing from the main body of text, consider adding it) Does your paper address subitem 1b-iii? Copy and paste relevant sections from the manuscript abstract (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or brie§y explain why the item is not applicable/relevant for your study Your answer 1b-v) CONCLUSIONS/DISCUSSION in abstract for negative trials Conclusions/Discussions in abstract for negative trials: Discuss the primary outcome - if the trial is negative (primary outcome not changed), and the intervention was not used, discuss whether negative results are attributable to lack of uptake and discuss reasons. (Note: Only report in the abstract what the main paper is reporting. If this information is missing from the main body of text, consider adding it) subitem not at all important 1 2 3 4 5 essential subitem not at all important 1 2 3 4 5 essential Does your paper address subitem 1b-iv? y p p Copy and paste relevant sections from the manuscript abstract (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or brie§y explain why the item is not applicable/relevant for your study Your answer Your answer https://docs.google.com/forms/d/e/1FAIpQLSfZBSUp1bwOc_OimqcS64RdfIAFvmrTSkZQL2-3O8O9hrL5Sw/viewform?hl=en_US&formkey=dGl… 11/47 https://docs.google.com/forms/d/e/1FAIpQLSfZBSUp1bwOc_OimqcS64RdfIAFvmrTSkZQL2-3O8O9hrL5Sw/viewform? m/forms/d/e/1FAIpQLSfZBSUp1bwOc_OimqcS64RdfIAFvmrTSkZQL2-3O8O9hrL5Sw/viewform?hl=en_US&formkey=dG 7/17/2019 CONSORT-EHEALTH (V 1.6.1) - Submission/Publication Form Does your paper address subitem 1b-v? Copy and paste relevant sections from the manuscript abstract (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or brie§y explain why the item is not applicable/relevant for your study Your answer Your answer INTRODUCTION 2a) In INTRODUCTION: Scienti¦c background and explanation of rationale 2a-i) Problem and the type of system/solution ) yp y Describe the problem and the type of system/solution that is object of the study: intended as stand- alone intervention vs. incorporated in broader health care program? Intended for a particular patient population? Goals of the intervention, e.g., being more cost-effective to other interventions, replace or complement other solutions? (Note: Details about the intervention are provided in “Methods” under 5) subitem not at all important 1 2 3 4 5 essential subitem not at all important 1 2 3 4 5 essential subitem not at all important m/forms/d/e/1FAIpQLSfZBSUp1bwOc_OimqcS64RdfIAFvmrTSkZQL2-3O8O9hrL5Sw/viewform?hl=en_US&formkey=dG ps://docs.google.com/forms/d/e/1FAIpQLSfZBSUp1bwOc_OimqcS64RdfIAFvmrTSkZQL2-3O8O9hrL5Sw/viewform?hl=e 7/17/2019 CONSORT-EHEALTH (V 1.6.1) - Submission/Publication Form Does your paper address subitem 2a-i? * Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or brie§y explain why the item is not applicable/relevant for your study Cross-cultural studies show high rates of depressive symptoms among students.However, as in many other Low income countries, there are barriers for accessing mental health treatments, such as cost and coverage, which prevent people from accessing the treatments they need. Internet-delivered treatments may be a suitable alternative to make evidence-based treatments available, especially when nearly 56% of the population in Colombia have access to internet. This study aimed at evaluating the e¨cacy of a culturally adapted cognitive-behavioural internet-delivered intervention (iCBT) Yo puedo sentirme bien/I can feel better programme for college students with depressive symptoms. This study is the ¦rst contribution to investigating the effectiveness of an iCBT in Colombia. 2a-ii) Scienti¦c background, rationale: What is known about the (type of) system Scienti¦c background, rationale: What is known about the (type of) system that is the object of the study (be sure to discuss the use of similar systems for other conditions/diagnoses, if appropiate), motivation for the study, i.e. what are the reasons for and what is the context for this speci¦c study, from which stakeholder viewpoint is the study performed, potential impact of ¦ndings [2]. Brie§y justify the choice of the comparator. subitem not at all important 1 2 3 4 5 essential https://docs.google.com/forms/d/e/1FAIpQLSfZBSUp1bwOc_OimqcS64RdfIAFvmrTSkZQL2-3O8O9hrL5Sw/viewform? 1FAIpQLSfZBSUp1bwOc_OimqcS64RdfIAFvmrTSkZQL2-3O8O9hrL5Sw/viewform?hl=en_US&formkey=dG… 13/47 7/17/2019 CONSORT-EHEALTH (V 1.6.1) - Submission/Publication Form Does your paper address CONSORT subitem 2b? * y p p Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like th to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or brie§y explain why the item is not applicable/relevant for your study This study aimed at evaluating the e¨cacy of a culturally adapted cognitive- behavioural internet-delivered intervention (iCBT) Yo puedo sentirme bien/I can feel better programme for college students with depressive symptoms. This study is the ¦rst contribution to investigating the effectiveness of an iCBT in Colombia. Does your paper address subitem 2a-ii? * y Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or brie§y explain why the item is not applicable/relevant for your study "Depressive disorders have shown to be more frequent among college students in comparison to the general population [15].". "Studies have shown that Colombia reports the second highest prevalence of major depression in Latin- America after Brazil [3, 4, 8]. ". "Despite the necessity to implement psychological interventions for depression in Latin America, there are only a few studies in this ¦eld [28]. More so, treatments are mostly pharmacological, and evidence-based psychological treatments are rarely used"."Research has found [39] that psychological treatments can enhance positive affect and decrease negative affect in depression, contributing to better outcomes". "Systematic review and meta-analysis have showed that Cognitive Behaviour Therapy (CBT) has been extensively researched and it has been demonstrated to be effective for depression [40, 41]."."Low-intensity internet-delivered treatment usually is based on cognitive-behavioural principles". 2b) In INTRODUCTION: Speci¦c objectives or hypotheses METHODS 3a) Description of trial design (such as parallel, factorial) including allocation ratio 3a) Description of trial design (such as parallel, factorial) including allocation ratio https://docs.google.com/forms/d/e/1FAIpQLSfZBSUp1bwOc_OimqcS64RdfIAFvmrTSkZQL2-3O8O9hrL5Sw/viewform?hl=en_US&formkey=dG… 14/47 https://docs.google.com/forms/d/e/1FAIpQLSfZBSUp1bwOc_OimqcS64RdfIAFvmrTSkZQL2-3O8O9hrL5Sw/viewform? m/forms/d/e/1FAIpQLSfZBSUp1bwOc_OimqcS64RdfIAFvmrTSkZQL2-3O8O9hrL5Sw/viewform?hl=en_US&formkey=dG 7/17/2019 CONSORT-EHEALTH (V 1.6.1) - Submission/Publication Form 3b) Important changes to methods after trial commencement (such as eligibility criteria), with reasons subitem not at all important 1 2 3 4 5 essential Does your paper address CONSORT subitem 3b? * Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or brie§y explain why the item is not applicable/relevant for your study It was not changes to methods after trial commencement. 3b-i) Bug ¦xes, Downtimes, Content Changes Bug ¦xes, Downtimes, Content Changes: ehealth systems are often dynamic systems. A description of changes to methods therefore also includes important changes made on the intervention or comparator during the trial (e.g., major bug ¦xes or changes in the functionality or content) (5-iii) and other “unexpected events” that may have in§uenced study design such as staff changes, system failures/downtimes, etc. [2]. Does your paper address subitem 3b-i? Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or brie§y explain why the item is not applicable/relevant for your study The Colombian iCBT programme for depression assigned as treatment in this study (Yo puedo sentirme bien / I can feel better) is a modi¦ed version of the clinically e¨cacious Space from Depression programme [56] used in Ireland and the United Kingdom. The programme was modi¦ed before to start the trial. 3b) Important changes to methods after trial commencement (such as eligibility criteria), with reasons Does your paper address CONSORT subitem 3a? * Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or brie§y explain why the item is not applicable/relevant for your study Does your paper address CONSORT subitem 3b? * Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or brie§y explain why the item is not applicable/relevant for your study It was not changes to methods after trial commencement. 3b-i) Bug ¦xes, Downtimes, Content Changes Bug ¦xes, Downtimes, Content Changes: ehealth systems are often dynamic systems. A description of changes to methods therefore also includes important changes made on the intervention or comparator during the trial (e.g., major bug ¦xes or changes in the functionality or content) (5-iii) and other “unexpected events” that may have in§uenced study design such as staff changes, system failures/downtimes, etc. [2]. subitem not at all important 1 2 3 4 5 essential Does your paper address subitem 3b-i? Design The study is a randomised control design examining the e¨cacy of the culturally-adapted intervention, in which participants were randomly assigned to two groups: a) culturally-adapted iCBT group and b) waiting list control group. Does your paper address subitem 3b-i? y p p Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or brie§y explain why the item is not applicable/relevant for your study The Colombian iCBT programme for depression assigned as treatment in this study (Yo puedo sentirme bien / I can feel better) is a modi¦ed version of the clinically e¨cacious Space from Depression programme [56] used in Ireland and the United Kingdom. The programme was modi¦ed before to start the trial. m/forms/d/e/1FAIpQLSfZBSUp1bwOc_OimqcS64RdfIAFvmrTSkZQL2-3O8O9hrL5Sw/viewform?hl=en_US&formkey=dG https://docs.google.com/forms/d/e/1FAIpQLSfZBSUp1bwOc_OimqcS64RdfIAFvmrTSkZQL2-3O8O9hrL5Sw/viewform?hl=en_US&formkey=dG… 15/47 7/17/2019 CONSORT-EHEALTH (V 1.6.1) - Submission/Publication Form 4a) Eligibility criteria for participants Does your paper address CONSORT subitem 4a? * Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or brie§y explain why the item is not applicable/relevant for your study subitem not at all important 1 2 3 4 5 essential information not in the ms, or brie§y explain why the item is not applicable/relevant for your study Eligibility Criteria -Exclusion criteria: Severe depressive symptoms >19 on PHQ-9 Suicidal ideation or intent: Score of 2 or above on PHQ-9 question 9 Psychosis Currently in psychological treatment for depression On medication for less than 1 month Alcohol or drugs misuse Previous diagnosis of an organic mental health disorder Depression preceding or coinciding a diagnosed medical condition - Inclusion criteria 18-year-old Mild to moderately severe depressive symptoms: (PHQ-9 score 10-19) 4a-i) Computer / Internet literacy Computer / Internet literacy is often an implicit “de facto” eligibility criterion - this should be explicitly clari¦ed. Does your paper address subitem 4a-i? Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or brie§y explain why the item is not applicable/relevant for your study Your answer Does your paper address subitem 4a-i? Does your paper address subitem 4a-i? y opy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" indicate direct quotes from your manuscript), or elaborate on this item by providing additional formation not in the ms, or brie§y explain why the item is not applicable/relevant for your study Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or brie§y explain why the item is not applicable/relevant for your study https://docs.google.com/forms/d/e/1FAIpQLSfZBSUp1bwOc_OimqcS64RdfIAFvmrTSkZQL2-3O8O9hrL5Sw/viewform?hl=en_US&formkey=dG… 16/47 m/forms/d/e/1FAIpQLSfZBSUp1bwOc_OimqcS64RdfIAFvmrTSkZQL2-3O8O9hrL5Sw/viewform?hl=en_US&formkey=dG https://docs.google.com/forms/d/e/1FAIpQLSfZBSUp1bwOc_OimqcS64RdfIAFvmrTSkZQL2-3O8O9hrL5Sw/viewform? CONSORT-EHEALTH (V 1.6.1) - Submission/Publication Form 7/17/2019 4a-ii) Open vs. closed, web-based vs. face-to-face assessments: Does your paper address subitem 4a-ii? * Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or brie§y explain why the item is not applicable/relevant for your study Participants received an email with an invitation to participate in the study. Once participants read the study information, informed consent was obtained from each user before screening and randomisation. Through the SilverCloud platform, participants were instructed to type their name on the informed consent page, to indicate that they had read and understood the study information and agreed to participate in the study. Thereafter, participants completed measures for screening purposes, including the Patient Health Questionnaire (PHQ-9), Sociodemographic & Clinical History Questionnaire and Generalised Anxiety Disorder-7 (GAD-7) questionnaire. Thereafter participants who were eligible for the study were randomised through computer algorithms and assigned into two groups the iCBT treatment group and waiting-list (WL) control group. a-ii) Open vs. closed, web-based vs. face-to-face assessments: 4a-ii) Open vs. closed, web-based vs. face-to-face assessments: Open vs. closed, web-based vs. face-to-face assessments: Mention how participants were recruited (online vs. o©ine), e.g., from an open access website or from a clinic, and clarify if this was a purely web-based trial, or there were face-to-face components (as part of the intervention or for assessment), i.e., to what degree got the study team to know the participant. In online-only trials, clarify if participants were quasi-anonymous and whether having multiple identities was possible or whether technical or logistical measures (e.g., cookies, email con¦rmation, phone calls) were used to detect/prevent these. subitem not at all important 1 2 3 4 5 essential subitem not at all important 1 2 3 4 5 essential 4a-iii) Information giving during recruitment 4a iii) Information giving during recruitment Information given during recruitment. Specify how participants were briefed for recruitment and in the informed consent procedures (e.g., publish the informed consent documentation as appendix, see also item X26), as this information may have an effect on user self-selection, user expectation and may also bias results. subitem not at all important 1 2 3 4 5 essential subitem not at all important subitem not at all important subitem not at all important essential https://docs.google.com/forms/d/e/1FAIpQLSfZBSUp1bwOc_OimqcS64RdfIAFvmrTSkZQL2-3O8O9hrL5Sw/viewform?hl=en_US&formkey=dG… 17/47 m/forms/d/e/1FAIpQLSfZBSUp1bwOc_OimqcS64RdfIAFvmrTSkZQL2-3O8O9hrL5Sw/viewform?hl=en_US&formkey=dG https://docs.google.com/forms/d/e/1FAIpQLSfZBSUp1bwOc_OimqcS64RdfIAFvmrTSkZQL2-3O8O9hrL5Sw/viewform? 7/17/2019 CONSORT-EHEALTH (V 1.6.1) - Submission/Publication Form Does your paper address subitem 4a-iii? Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or brie§y explain why the item is not applicable/relevant for your study An email was sent to all college students. Potential participants were able to visit a website to receive information about the study, participation criteria, information about the treatment and how to get in contact to proceed with the study. 4b) Settings and locations where the data were collected 4b) Settings and locations where the data were collected Does your paper address CONSORT subitem 4b? * Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or brie§y explain why the item is not applicable/relevant for your study College students from two cities and universities in Colombia were included in this study. Both undergraduate and graduate level students from any school at a university in Bogota city and students of psychology, medicine, nursing and education from a university in Bucaramanga city in Colombia were eligible to participate Clearly report if outcomes were (self-)assessed through online questionnaires (as common in we based trials) or otherwise. subitem not at all important 1 2 3 4 5 essential Clearly report if outcomes were (self-)assessed through online questionnaires (as common in web- based trials) or otherwise. subitem not at all important 1 2 3 4 5 essential Does your paper address subitem 4b-i? * 4b-ii) Report how institutional a¨liations are displayed Report how institutional a¨liations are displayed to potential participants [on ehealth media], as a¨liations with prestigious hospitals or universities may affect volunteer rates, use, and reactions with regards to an intervention.(Not a required item – describe only if this may bias results) subitem not at all important 1 2 3 4 5 essential subitem not at all important 1 2 3 4 5 essential subitem not at all important 1 2 3 4 5 essential subitem not at all important 1 2 3 4 5 essential Does your paper address subitem 4b-ii? Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or brie§y explain why the item is not applicable/relevant for your study Your answer 5) The interventions for each group with su¨cient details to allow replication, including how and when they were actually administered 5-i) Mention names, credential, a¨liations of the developers, sponsors, and owners Mention names, credential, a¨liations of the developers, sponsors, and owners [6] (if authors/evaluators are owners or developer of the software, this needs to be declared in a “Con of interest” section or mentioned elsewhere in the manuscript). Does your paper address subitem 4b-i? * y p p Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or brie§y explain why the item is not applicable/relevant for your study Through the SilverCloud platform participants completed measures for screening purposes, including the Patient Health Questionnaire (PHQ-9), Sociodemographic & Clinical History Questionnaire and Generalised Anxiety Disorder-7 (GAD-7) questionnaire. https://docs.google.com/forms/d/e/1FAIpQLSfZBSUp1bwOc_OimqcS64RdfIAFvmrTSkZQL2-3O8O9hrL5Sw/viewform?hl=en_US&formkey=dG… 18/47 m/forms/d/e/1FAIpQLSfZBSUp1bwOc_OimqcS64RdfIAFvmrTSkZQL2-3O8O9hrL5Sw/viewform?hl=en_US&formkey=dG https://docs.google.com/forms/d/e/1FAIpQLSfZBSUp1bwOc_OimqcS64RdfIAFvmrTSkZQL2-3O8O9hrL5Sw/viewform? 7/17/2019 CONSORT-EHEALTH (V 1.6.1) - Submission/Publication Form Does your paper address subitem 5-ii? Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or brie§y explain why the item is not applicable/relevant for your study Your answer 5-ii) Describe the history/development process Describe the history/development process of the application and previous formative evaluations (e.g., focus groups, usability testing), as these will have an impact on adoption/use rates and help with interpreting results. subitem not at all important 1 2 3 4 5 essential Does your paper address subitem 5-i? Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or brie§y explain why the item is not applicable/relevant for your study Your answer https://docs.google.com/forms/d/e/1FAIpQLSfZBSUp1bwOc_OimqcS64RdfIAFvmrTSkZQL2-3O8O9hrL5Sw/viewform? m/forms/d/e/1FAIpQLSfZBSUp1bwOc_OimqcS64RdfIAFvmrTSkZQL2-3O8O9hrL5Sw/viewform?hl=en_US&formkey=dG 7/17/2019 CONSORT-EHEALTH (V 1.6.1) - Submission/Publication Form 5-iii) Revisions and updating Revisions and updating. Clearly mention the date and/or version number of the application/intervention (and comparator, if applicable) evaluated, or describe whether the intervention underwent major changes during the evaluation process, or whether the development and/or content was “frozen” during the trial. Describe dynamic components such as news feeds or changing content which may have an impact on the replicability of the intervention (for unexpected events see item 3b). subitem not at all important 1 2 3 4 5 essential subitem not at all important 1 2 3 4 5 essential 7/17/2019 CONSORT-EHEALTH (V 1.6.1) - Submission/Publication Form subitem not at all important 1 2 3 4 5 essential subitem not at all important 1 2 3 4 5 essential Does your paper address subitem 5-iv? Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or brie§y explain why the item is not applicable/relevant for your study At the beginning of each session participants were asked to re§ect on their previous session and complete the Helpful Aspects of Treatment Form (HAT) a qualitative measure, the results of which are not presented in this paper. 5-v) Ensure replicability by publishing the source code, and/or providing screenshots/screen-capture video, and/or providing §owcharts of the algorithms used Ensure replicability by publishing the source code, and/or providing screenshots/screen-capture video, and/or providing §owcharts of the algorithms used. Replicability (i.e., other researchers should in principle be able to replicate the study) is a hallmark of scienti¦c reporting. Does your paper address subitem 5-v? Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or brie§y explain why the item is not applicable/relevant for your study Your answer 5-vi) Digital preservation Digital preservation: Provide the URL of the application, but as the intervention is likely to change or disappear over the course of the years; also make sure the intervention is archived (Internet Archive, webcitation.org, and/or publishing the source code or screenshots/videos alongside the article). As pages behind login screens cannot be archived, consider creating demo pages which are accessible without login. Does your paper address subitem 5-vi? Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or brie§y explain why the item is not applicable/relevant for your study 7/17/2019 CONSORT-EHEALTH (V 1.6.1) - Submission/Publication Form 5-v) Ensure replicability by publishing the source code, and/or providing screenshots/screen-capture video, and/or providing §owcharts of the algorithms used Ensure replicability by publishing the source code, and/or providing screenshots/screen-capture video, and/or providing §owcharts of the algorithms used. Replicability (i.e., other researchers should in principle be able to replicate the study) is a hallmark of scienti¦c reporting. g Ensure replicability by publishing the source code, and/or providing screenshots/screen-capture video, and/or providing §owcharts of the algorithms used. Replicability (i.e., other researchers should in principle be able to replicate the study) is a hallmark of scienti¦c reporting. subitem not at all important 1 2 3 4 5 essential subitem not at all important 1 2 3 4 5 essential Does your paper address subitem 5-v? 5-vi) Digital preservation Digital preservation: Provide the URL of the application, but as the intervention is likely to change or disappear over the course of the years; also make sure the intervention is archived (Internet Archive, webcitation.org, and/or publishing the source code or screenshots/videos alongside the article). As pages behind login screens cannot be archived, consider creating demo pages which are accessible without login. subitem not at all important 1 2 3 4 5 essential Does your paper address subitem 5-v? Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or brie§y explain why the item is not applicable/relevant for your study Your answer Does your paper address subitem 5-iv? Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or brie§y explain why the item is not applicable/relevant for your study At the beginning of each session participants were asked to re§ect on their previous session and complete the Helpful Aspects of Treatment Form (HAT) a qualitative measure, the results of which are not presented in this paper. 5-v) Ensure replicability by publishing the source code, and/or providing screenshots/screen-capture video, and/or providing §owcharts of the algorithms used Ensure replicability by publishing the source code, and/or providing screenshots/screen-capture video, and/or providing §owcharts of the algorithms used. Replicability (i.e., other researchers should in principle be able to replicate the study) is a hallmark of scienti¦c reporting. Does your paper address subitem 5-iii? Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or brie§y explain why the item is not applicable/relevant for your study The cultural adaptation that underwent several steps is described in a separate study [57]. The adaptation involved three distinct phases based on an integrative approach proposed by the author (AS). https://docs.google.com/forms/d/e/1FAIpQLSfZBSUp1bwOc_OimqcS64RdfIAFvmrTSkZQL2-3O8O9hrL5Sw/viewform?hl=en_US&formkey=dG… 20/47 Does your paper address subitem 5-vi? Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or brie§y explain why the item is not applicable/relevant for your study Your answer Your answer https://docs.google.com/forms/d/e/1FAIpQLSfZBSUp1bwOc_OimqcS64RdfIAFvmrTSkZQL2-3O8O9hrL5Sw/viewform? 7/17/2019 CONSORT-EHEALTH (V 1.6.1) - Submission/Publication Form Does your paper address subitem 5-vii? * Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or brie§y explain why the item is not applicable/relevant for your study "An email with information of the study and a link to access to the treatment was sent to all college students". "The participants accessed the intervention for free during the time of the study was carried out. Also, the participants had access to the programme without a supporter a year after ¦nishing the study if they wanted to use it". 5-vii) Access Access: Describe how participants accessed the application, in what setting/context, if they had to pay (or were paid) or not, whether they had to be a member of speci¦c group. If known, describe how participants obtained “access to the platform and Internet” [1]. To ensure access for editors/reviewers/readers, consider to provide a “backdoor” login account or demo mode for reviewers/readers to explore the application (also important for archiving purposes, see vi). subitem not at all important 1 2 3 4 5 essential subitem not at all important 1 2 3 4 5 essential 5-viii) Mode of delivery, features/functionalities/components of the intervention and comparator, and the theoretical framework Describe mode of delivery, features/functionalities/components of the intervention and comparator, and the theoretical framework [6] used to design them (instructional strategy [1], behaviour change techniques, persuasive features, etc., see e.g., [7, 8] for terminology). This includes an in-depth description of the content (including where it is coming from and who developed it) [1],” whether [and how] it is tailored to individual circumstances and allows users to track their progress and receive feedback” [6]. This also includes a description of communication delivery channels and – if computer-mediated communication is a component – whether communication was synchronous or asynchronous [6]. It also includes information on presentation strategies [1], including page design principles, average amount of text on pages, presence of hyperlinks to other resources, etc. [1]. subitem not at all important 1 2 3 4 5 essential subitem not at all important 1 2 3 4 5 essential subitem not at all important subitem not at all important https://docs.google.com/forms/d/e/1FAIpQLSfZBSUp1bwOc_OimqcS64RdfIAFvmrTSkZQL2-3O8O9hrL5Sw/viewform? m/forms/d/e/1FAIpQLSfZBSUp1bwOc_OimqcS64RdfIAFvmrTSkZQL2-3O8O9hrL5Sw/viewform?hl=en_US&formkey=dG 7/17/2019 CONSORT-EHEALTH (V 1.6.1) - Submission/Publication Form Does your paper address subitem 5-ix? Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or brie§y explain why the item is not applicable/relevant for your study Your answer 5-ix) Describe use parameters Describe use parameters (e.g., intended “doses” and optimal timing for use). Clarify what instructions or recommendations were given to the user, e.g., regarding timing, frequency, heaviness of use, if any, or was the intervention used ad libitum. subitem not at all important 1 2 3 4 5 essential subitem not at all important 1 2 3 4 5 essential Does your paper address subitem 5-viii? * Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or brie§y explain why the item is not applicable/relevant for your study Both Yo puedo sentirme bien (the Colombian iCBT programme used as treatment in this study) and Space from depression (the original programme from which it was culturally and linguistically adapted) consist of seven modules of cognitive-behavioural therapy (Table 2). The treatment includes self- monitoring, behavioural activation, cognitive restructuring, and challenging core beliefs. All modules have the same structure and format, which consist of quizzes, videos, educational content, activities with homework suggestions and a module review page. Also, users have a supporter, who gives a weekly feedback to the user asynchronously [56]. 5-x) Clarify the level of human involvement ) y Clarify the level of human involvement (care providers or health professionals, also technical assistance) in the e-intervention or as co-intervention (detail number and expertise of professionals involved, if any, as well as “type of assistance offered, the timing and frequency of the support, how it is initiated, and the medium by which the assistance is delivered”. It may be necessary to distinguish between the level of human involvement required for the trial, and the level of human involvement required for a routine application outside of a RCT setting (discuss under item 21 – generalizability). subitem not at all important https://docs.google.com/forms/d/e/1FAIpQLSfZBSUp1bwOc_OimqcS64RdfIAFvmrTSkZQL2-3O8O9hrL5Sw/viewform? 7/17/2019 CONSORT-EHEALTH (V 1.6.1) - Submission/Publication Form Does your paper address subitem 5-x? Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or brie§y explain why the item is not applicable/relevant for your study Your answer 5-xi) Report any prompts/reminders used 5-xi) Report any prompts/reminders used Report any prompts/reminders used: Clarify if there were prompts (letters, emails, phone calls, SMS) to use the application, what triggered them, frequency etc. It may be necessary to distinguish between the level of prompts/reminders required for the trial, and the level of prompts/reminders for a routine application outside of a RCT setting (discuss under item 21 – generalizability). subitem not at all important 1 2 3 4 5 essential subitem not at all important 1 2 3 4 5 essential subitem not at all important Does your paper address subitem 5-xi? * Does your paper address subitem 5-xi? * Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or brie§y explain why the item is not applicable/relevant for your study Also, participants received emails and calls by the research team at the post- treatment and 3-month follow up time points to encourage them to complete their assigned measures. 5-xii) Describe any co-interventions (incl. training/support) 5-xii) Describe any co-interventions (incl. training/support) Describe any co-interventions (incl. training/support): Clearly state any interventions that are provided in addition to the targeted eHealth intervention, as ehealth intervention may not be designed as stand-alone intervention. This includes training sessions and support [1]. It may be necessary to distinguish between the level of training required for the trial, and the level of training for a routine application outside of a RCT setting (discuss under item 21 – generalizability. subitem not at all important 1 2 3 4 5 essential subitem not at all important 1 2 3 4 5 essential subitem not at all important 1 2 3 4 5 essential subitem not at all important subitem not at all important essential m/forms/d/e/1FAIpQLSfZBSUp1bwOc_OimqcS64RdfIAFvmrTSkZQL2-3O8O9hrL5Sw/viewform?hl=en_US&formkey=dG https://docs.google.com/forms/d/e/1FAIpQLSfZBSUp1bwOc_OimqcS64RdfIAFvmrTSkZQL2-3O8O9hrL5Sw/viewform? 7/17/2019 CONSORT-EHEALTH (V 1.6.1) - Submission/Publication Form Does your paper address subitem 5-xii? * Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or brie§y explain why the item is not applicable/relevant for your study Each of the 214 participants were assigned a supporter who was a postgraduate student in clinical psychology with supervised experience in CBT with adults. Ten supporters were trained by the principal researcher (AS) in using the Yo puedo sentirme bien / I can feel better platform and programme before starting their role as a supporter and they were supervised by an experienced clinical psychologist at the university. 6a) Completely de¦ned pre-speci¦ed primary and secondary outcome measures, including how and when they were assessed Does your paper address CONSORT subitem 6a? * Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or brie§y explain why the item is not applicable/relevant for your study Through the SilverCloud platform the assessments were completed by the participants. Participants were assessed at baseline, post-treatment and follow- up through the Patient Health Questionnaire (PHQ-9) and Generalised Anxiety Disorder-7 (GAD-7). Primary outcome: PHQ-9 [62] and Secondary outcome measure GAD-7[66]. The assessment started in August 2016. 6a-i) Online questionnaires: describe if they were validated for online use and apply CHERRIES items to describe how the questionnaires were designed/deployed If outcomes were obtained through online questionnaires, describe if they were validated for online use and apply CHERRIES items to describe how the questionnaires were designed/deployed [9]. 6a-i) Online questionnaires: describe if they were validated for online use and apply CHERRIES items to describe how the questionnaires were designed/deployed If outcomes were obtained through online questionnaires, describe if they were validated for online use and apply CHERRIES items to describe how the questionnaires were designed/deployed [9]. subitem not at all important 1 2 3 4 5 essential Your answer 7/17/2019 CONSORT-EHEALTH (V 1.6.1) - Submission/Publication Form ) Describe whether and how “use” (including intensity of use/dosage) was de¦ned/measured/monitored (logins, log¦le analysis, etc.). Use/adoption metrics are important process outcomes that should be reported in any ehealth trial. Does your paper address subitem 6a-ii? Copy and paste relevant sections from manuscript text A session in an online intervention is de¦ned as an instance when a client logged into the system [56]. Session time estimation is not exact because users may be interrupted or take breaks within session, and may not formally log out of the system. 6a-iii) Describe whether, how, and when qualitative feedback from participants was obtained Describe whether, how, and when qualitative feedback from participants was obtained (e.g., through emails, feedback forms, interviews, focus groups). Does your paper address subitem 6a-iii? Copy and paste relevant sections from manuscript text At post-treatment, participants were asked to complete a measure of satisfaction with treatment (SAT). SAT asks users about positive and negative experiences with the internet-delivered treatment. SAT contains two questions asking to describe what participants most liked and least liked about the online treatment 6b) Any changes to trial outcomes after the trial commenced, with reasons https://docs.google.com/forms/d/e/1FAIpQLSfZBSUp1bwOc_OimqcS64RdfIAFvmrTSkZQL2-3O8O9hrL5Sw/viewform?hl=en_US&formkey=dG… 26/47 1FAIpQLSfZBSUp1bwOc_OimqcS64RdfIAFvmrTSkZQL2-3O8O9hrL5Sw/viewform?hl=en_US&formkey=dG… 26/47 https://docs.google.com/forms/d/e/1FAIpQLSfZBSUp1bwOc_OimqcS64RdfIAFvmrTSkZQL2-3O8O9hrL5Sw/viewform? 7/17/2019 CONSORT-EHEALTH (V 1.6.1) - Submission/Publication Form 7a) How sample size was determined NPT: When applicable, details of whether and how the clustering by care provides or centers was addressed subitem not at all important 1 2 3 4 5 essential 7b) When applicable, explanation of any interim analyses and stopping guidelines py p p ( q q to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or brie§y explain why the item is not applicable/relevant for your study Due to the very high percentage (80%, n= 86) of missing data in the sample, we decided not to follow the protocol data analysis strategy [60]. Instead we modi¦ed our approach include only those participants who provided post- treatment measures (20%, n=21) and/or 3- months follow up (17%, n=18) measures. 7a-i) Describe whether and how expected attrition was taken into account when calculating the sample size Describe whether and how expected attrition was taken into account when calculating the sample size. Does your paper address subitem 7a-i? Copy and paste relevant sections from manuscript title (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or brie§y explain why the item is not applicable/relevant for your study Your answer Due to the very high percentage (80%, n= 86) of missing data in the sample, we decided not to follow the protocol data analysis strategy [60]. Instead we modi¦ed our approach include only those participants who provided post- treatment measures (20%, n=21) and/or 3- months follow up (17%, n=18) measures. 7a) How sample size was determined 7a-i) Describe whether and how expected attrition was taken into account when calculating the sample size Describe whether and how expected attrition was taken into account when calculating the sample size. Does your paper address CONSORT subitem 6b? * Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or brie§y explain why the item is not applicable/relevant for your study Does your paper address subitem 7a-i? y p p Copy and paste relevant sections from manuscript title (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or brie§y explain why the item is not applicable/relevant for your study Your answer Your answer 7b) When applicable, explanation of any interim analyses and stopping guidelines 1FAIpQLSfZBSUp1bwOc_OimqcS64RdfIAFvmrTSkZQL2-3O8O9hrL5Sw/viewform?hl=en_US&formkey=dG… 27/47 https://docs.google.com/forms/d/e/1FAIpQLSfZBSUp1bwOc_OimqcS64RdfIAFvmrTSkZQL2-3O8O9hrL5Sw/viewform? 7/17/2019 CONSORT-EHEALTH (V 1.6.1) - Submission/Publication Form 8a) Method used to generate the random allocation sequence NPT: When applicable, how care providers were allocated to each trial group 8b) Type of randomisation; details of any restriction (such as blocking and block size) 9) Mechanism used to implement the random allocation sequence (such as sequentially numbered containers), describing any steps taken to conceal the sequence until interventions were assigned and/or 3- months follow up measures. Does your paper address CONSORT subitem 8a? * Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or brie§y explain why the item is not applicable/relevant for your study Randomisation was handled by a computer algorithm administered by a person independent of the researchers. The randomisation was 1:1 Does your paper address CONSORT subitem 8b? * Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or brie§y explain why the item is not applicable/relevant for your study Post baseline, screened eligible participants were randomised and informed immediately about their group assignment.The randomisation was blocked when the number of users were achieved. 8a) Method used to generate the random allocation sequence NPT: When applicable, how care providers were allocated to each trial group Does your paper address CONSORT subitem 8a? * Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or brie§y explain why the item is not applicable/relevant for your study Randomisation was handled by a computer algorithm administered by a person independent of the researchers. The randomisation was 1:1 8b) Type of randomisation; details of any restriction (such as blocking and block size) Does your paper address CONSORT subitem 7b? * Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or brie§y explain why the item is not applicable/relevant for your study It was not any interim analysis. Due to the very high percentage of missing data in the sample, we not to follow the protocol data analysis strategy [60]. Instead we include only those participants who provided post-treatment measures and/or 3- months follow up measures. Does your paper address CONSORT subitem 8b? * Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or brie§y explain why the item is not applicable/relevant for your study Post baseline, screened eligible participants were randomised and informed immediately about their group assignment.The randomisation was blocked when the number of users were achieved. 9) Mechanism used to implement the random allocation sequence (such as sequentially numbered containers), describing any steps taken to conceal the sequence until interventions were assigned https://docs.google.com/forms/d/e/1FAIpQLSfZBSUp1bwOc_OimqcS64RdfIAFvmrTSkZQL2-3O8O9hrL5Sw/viewform?hl=en_US&formkey=dG… 28/47 1FAIpQLSfZBSUp1bwOc_OimqcS64RdfIAFvmrTSkZQL2-3O8O9hrL5Sw/viewform?hl=en_US&formkey=dG… 28/47 ogle.com/forms/d/e/1FAIpQLSfZBSUp1bwOc_OimqcS64RdfIAFvmrTSkZQL2-3O8O9hrL5Sw/viewform?hl=en_US&form 7/17/2019 CONSORT-EHEALTH (V 1.6.1) - Submission/Publication Form Does your paper address CONSORT subitem 9? * Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or brie§y explain why the item is not applicable/relevant for your study An email with information of the study and a link to access to the treatment was sent to all college students. Once participants read the study information, informed consent was obtained from each user before screening and randomisation. Through the SilverCloud platform, participants were instructed to type their name on the informed consent page, to indicate that they had read and understood the study information and agreed to participate in the study. Thereafter participants who were eligible for the study were randomised through computer algorithms and assigned into two groups the iCBT treatment group and waiting-list (WL) control group. Those assigned to the active treatment started the internet-delivered treatment immediately for seven weeks, while the WL participants’ treatment started seven weeks later. Individuals not meeting the inclusion criteria at baseline assessment were referred to other appropriate sources of face-to-face support at the student counselling service in their respective universities. Once the students completed the questionnaires, they received immediate feedback on their results. If exclusion from the study was necessary, information and advice to see the counselling service at their college was provided to the student. 10) Who generated the random allocation sequence, who enrolled participants, and who assigned participants to interventions Does your paper address CONSORT subitem 10? * Does your paper address CONSORT subitem 10? * Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or brie§y explain why the item is not applicable/relevant for your study y p p Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like th to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or brie§y explain why the item is not applicable/relevant for your study Randomisation was handled by a computer algorithm administered by a person independent of the researchers 11a) If done, who was blinded after assignment to interventions (for example, participants, care providers, those assessing outcomes) and how NPT: Whether or not administering co-interventions were blinded to group assignment NPT: Whether or not administering co-interventions were blinded to group assignment CONSORT-EHEALTH (V 1.6.1) - Submission/Publication Form 7/17/2019 11a-i) Specify who was blinded, and who wasn’t Specify who was blinded, and who wasn’t. Usually, in web-based trials it is not possible to blind the participants [1, 3] (this should be clearly acknowledged), but it may be possible to blind outcome assessors, those doing data analysis or those administering co-interventions (if any). subitem not at all important 1 2 3 4 5 essential subitem not at all important 1 2 3 4 5 essential 11a-ii) Discuss e.g., whether participants knew which intervention was the “intervention of interest” and which one was the “comparator” Informed consent procedures (4a-ii) can create biases and certain expectations - discuss e.g., whether participants knew which intervention was the “intervention of interest” and which one was the “comparator”. Does your paper address subitem 11a-i? * y Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or brie§y explain why the item is not applicable/relevant for your study Each participant had feedback on their scores from the assessments. The participants were blinded from the assessments of other users. Supporters could see the scores only from participants who were assigned to him/her in the trial. Supporters were blinded of the questionnaire "Helpful and Hindering Aspects of Treatment (HAT)" outcomes. Only the research group had access to all the data analysis. Does your paper address subitem 11a-ii? Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or brie§y explain why the item is not applicable/relevant for your study Your answer 7/17/2019 CONSORT-EHEALTH (V 1.6.1) - Submission/Publication Form 12a-i) Imputation techniques to deal with attrition / missing values Imputation techniques to deal with attrition / missing values: Not all participants will use the intervention/comparator as intended and attrition is typically high in ehealth trials. Specify how participants who did not use the application or dropped out from the trial were treated in the statistical analysis (a complete case analysis is strongly discouraged, and simple imputation techniques such as LOCF may also be problematic [4]). subitem not at all important 1 2 3 4 5 essential 12a) Statistical methods used to compare groups for primary and secondary outcomes 12a) Statistical methods used to compare groups for primary and secondary outcomes NPT: When applicable, details of whether and how the clustering by care providers or centers was addressed Does your paper address CONSORT subitem 12a? * Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or brie§y explain why the item is not applicable/relevant for your study The effect of treatment on PHQ-9 and GAD-7 severity scores was evaluated separately for each measure using repeated-measures linear mixed-effects models ¦t with restricted maximum likelihood in the R package lme4. Does your paper address CONSORT subitem 11b? * Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or brie§y explain why the item is not applicable/relevant for your study N/A. The trial compared an active group to a waiting list control group Does your paper address subitem X26-i? y p p Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like th to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or brie§y explain why the item is not applicable/relevant for your study Your answer Does your paper address subitem 12a-i? * y p p Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or brie§y explain why the item is not applicable/relevant for your study Due to the very high percentage (80%, n= 86) of missing data in the sample, we decided not to follow the protocol data analysis strategy [60]. Instead we modi¦ed our approach include only those participants who provided post- treatment measures (20%, n=21) and/or 3- months follow up (17%, n=18) measures. https://docs.google.com/forms/d/e/1FAIpQLSfZBSUp1bwOc_OimqcS64RdfIAFvmrTSkZQL2-3O8O9hrL5Sw/viewform?hl=en_US&formkey=dG… 31/47 7/17/2019 CONSORT-EHEALTH (V 1.6.1) - Submission/Publication Form 12b) Methods for additional analyses, such as subgroup analyses and adjusted analyses Does your paper address CONSORT subitem 12b? * Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or brie§y explain why the item is not applicable/relevant for your study Descriptive statistics was used to analyse the quantitative data from the Satisfaction with treatment (SAT) questions and the qualitative responses from this questionnaire were analysed using thematic analysis. The categorisation of the data followed those identi¦ed in previous research [75]. subitem not at all important CONSORT-EHEALTH (V 1.6.1) - Submission/Publication Form subitem not at all important 1 2 3 4 5 essential RESULTS 13a) For each group, the numbers of participants who were randomly assigned, received intended treatment, and were analysed for the primary outcome NPT: The number of care providers or centers performing the intervention in each group and the number of patients treated by each care provider in each center Does your paper address subitem X26-ii? Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or brie§y explain why the item is not applicable/relevant for your study Your answer X26-iii) Safety and security procedures Safety and security procedures, incl. privacy considerations, and any steps taken to reduce the likelihood or detection of harm (e.g., education and training, availability of a hotline) Does your paper address subitem X26-iii? Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or brie§y explain why the item is not applicable/relevant for your study Your answer Does your paper address CONSORT subitem 13a? * Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional 7/17/2019 CONSORT-EHEALTH (V 1.6.1) - Submission/Publication Form RESULTS 13a) For each group, the numbers of participants who were randomly assigned, received intended treatment, and were analysed for the primary outcome NPT: The number of care providers or centers performing the intervention in each group and the number of patients treated by each care provider in each center X26-iii) Safety and security procedures X26-iii) Safety and security procedures Safety and security procedures, incl. privacy considerations, and any steps taken to reduce the likelihood or detection of harm (e.g., education and training, availability of a hotline) Safety and security procedures, incl. privacy considerations, and any steps taken to reduce the likelihood or detection of harm (e.g., education and training, availability of a hotline) Does your paper address subitem X26-iii? Does your paper address subitem X26-ii? Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or brie§y explain why the item is not applicable/relevant for your study Your answer Does your paper address subitem X26-iii? Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or brie§y explain why the item is not applicable/relevant for your study Your answer x26-ii) Outline informed consent procedures Outline informed consent procedures e.g., if consent was obtained o©ine or online (how? Checkbox, etc.?), and what information was provided (see 4a-ii). See [6] for some items to be included in informed consent documents. https://docs.google.com/forms/d/e/1FAIpQLSfZBSUp1bwOc_OimqcS64RdfIAFvmrTSkZQL2-3O8O9hrL5Sw/viewform?hl=en_US&formkey=dG… 32/47 13b) For each group, losses and exclusions after randomisation, together with reasons 13b) For each group, losses and exclusions after randomisation, together with reasons Does your paper address CONSORT subitem 13b? (NOTE: Preferably, this is shown in a CONSORT §ow diagram) * Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or brie§y explain why the item is not applicable/relevant for your study Of the 107 randomised to the immediate treatment group 80% (n=86) began module 1 and 9.3% (n=10) completed all modules. There was not exclusions after randomisation. 14a) Dates de¦ning the periods of recruitment and follow-up 14a) Dates de¦ning the periods of recruitment and follow-up 13b-i) Attrition diagram ) g Strongly recommended: An attrition diagram (e.g., proportion of participants still logging in or using the intervention/comparator in each group plotted over time, similar to a survival curve) or other ¦gures or tables demonstrating usage/dose/engagement. subitem not at all important 1 2 3 4 5 essential subitem not at all important 1 2 3 4 5 essential subitem not at all important 1 2 3 4 5 essential Does your paper address subitem 13b-i? Copy and paste relevant sections from the manuscript or cite the ¦gure number if applicable (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or brie§y explain why the item is not applicable/relevant for your study Your answer Your answer Does your paper address CONSORT subitem 13a? * Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or brie§y explain why the item is not applicable/relevant for your study 604 participants were screened, of which 390 were excluded based on the established criteria. A signi¦cant amount of participants were excluded due to suicidal ideation (153 individuals; 25%) and misuse of drugs or alcohol (116 students, 19%). At sign-up, there were a total of 107 participants assigned to the treatment group and 107 assigned to the waiting list group. m/forms/d/e/1FAIpQLSfZBSUp1bwOc_OimqcS64RdfIAFvmrTSkZQL2-3O8O9hrL5Sw/viewform?hl=en_US&formkey=dG https://docs.google.com/forms/d/e/1FAIpQLSfZBSUp1bwOc_OimqcS64RdfIAFvmrTSkZQL2-3O8O9hrL5Sw/viewform?hl=en_US&formkey=dG… 33/47 7/17/2019 CONSORT-EHEALTH (V 1.6.1) - Submission/Publication Form Does your paper address CONSORT subitem 14b? * Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or brie§y explain why the item is not applicable/relevant for your study The trial followed the time protocol method. The trial ended when all the measures were completed by the participants. 15) A table showing baseline demographic and clinical characteristics for each group NPT: When applicable, a description of care providers (case volume, quali¦cation, expertise, etc.) and centers (volume) in each group Does your paper address subitem 14a-i? Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or brie§y explain why the item is not applicable/relevant for your study Your answer Your answer 14b) Why the trial ended or was stopped (early) 14a-i) Indicate if critical “secular events” fell into the study period p Indicate if critical “secular events” fell into the study period, e.g., signi¦cant changes in Internet resources available or “changes in computer hardware or Internet delivery resources” CONSORT-EHEALTH (V 1.6.1) - Submission/Publication Form subitem not at all important 1 2 3 4 5 essential 14b) Why the trial ended or was stopped (early) 15) A table showing baseline demographic and clinical characteristics for each group 14a-i) Indicate if critical “secular events” fell into the study period Indicate if critical “secular events” fell into the study period, e.g., signi¦cant changes in Internet resources available or “changes in computer hardware or Internet delivery resources” Does your paper address subitem 14a-i? Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or brie§y explain why the item is not applicable/relevant for your study Your answer Does your paper address CONSORT subitem 14b? * Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or brie§y explain why the item is not applicable/relevant for your study The trial followed the time protocol method. The trial ended when all the measures were completed by the participants. CONSORT-EHEALTH (V 1.6.1) - Submission/Publication Form 7/17/2019 Does your paper address CONSORT subitem 14a? * Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or brie§y explain why the item is not applicable/relevant for your study The recruitment was in August 2016 and January 2017. Those participants assigned to the active treatment started the internet-delivered treatment immediately for seven weeks, while the WL participants’ treatment started seven weeks later. Also, participants completed measures at the post-treatment and 3- month follow up time points. https://docs.google.com/forms/d/e/1FAIpQLSfZBSUp1bwOc_OimqcS64RdfIAFvmrTSkZQL2-3O8O9hrL5Sw/viewform?hl=en_US&formkey=dG… 34/47 15-i) Report demographics associated with digital divide issues 15-i) Report demographics associated with digital divide issues In ehealth trials it is particularly important to report demographics associated with digital divide issues, such as age, education, gender, social-economic status, computer/Internet/ehealth literacy of the participants, if known. Does your paper address CONSORT subitem 15? * Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or brie§y explain why the item is not applicable/relevant for your study Descriptive statistics revealed that at post-randomisation, there were no signi¦cant differences in the sample between the iCBT group and waiting list control group (WL) on any variables. Table 4 shows details of the characteristics of the sample. https://docs.google.com/forms/d/e/1FAIpQLSfZBSUp1bwOc_OimqcS64RdfIAFvmrTSkZQL2-3O8O9hrL5Sw/viewform?hl=en_US&formkey=dG… 35/47 m/forms/d/e/1FAIpQLSfZBSUp1bwOc_OimqcS64RdfIAFvmrTSkZQL2-3O8O9hrL5Sw/viewform?hl=en_US&formkey=dG https://docs.google.com/forms/d/e/1FAIpQLSfZBSUp1bwOc_OimqcS64RdfIAFvmrTSkZQL2-3O8O9hrL5Sw/viewform? CONSORT-EHEALTH (V 1.6.1) - Submission/Publication Form 7/17/2019 Does your paper address subitem 15-i? * https://docs.google.com/forms/d/e/1FAIpQLSfZBSUp1bwOc_OimqcS64RdfIAFvmrTSkZQL2-3O8O9hrL5Sw/viewform?hl=en_US&formkey=dG… 36/47 7/17/2019 CONSORT-EHEALTH (V 1.6.1) - Submission/Publication Form Does your paper address subitem 15-i? * Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or brie§y explain why the item is not applicable/relevant for your study The mean age was 22.15 (S.D = 4.74). All of the participants were full time students. The majority of the college students sample was from health sciences (40%, n=86), followed by social sciences (29%, n=62) and Engineering (17%, n=37). There was no signi¦cant difference between the proportions of participants who reported a previous diagnosis of depression in the control group vs the treatment group. The majority of the participants reported depressive symptoms of between 1 and 2 years (35%, n=75). Twenty-two percent (22%, n=47) reported to have had counselling/psychotherapy for depression in the past. Lastly, the sample reported con¦dence in their use of information technology with 37% (n=72) of participants reporting feeling con¦dent. Forty one percent (41%, n=87)) of the participants reported feeling not so con¦dent using technology. https://docs.google.com/forms/d/e/1FAIpQLSfZBSUp1bwOc_OimqcS64RdfIAFvmrTSkZQL2-3O8O9hrL5Sw/viewform?hl=en_US&formkey=dG… 36/47 16) For each group, number of participants (denominator) included in each analysis and whether the analysis was by original assigned groups subitem not at all important 1 2 3 4 5 essential 16-i) Report multiple “denominators” and provide de¦nitions Report multiple “denominators” and provide de¦nitions: Report N’s (and effect sizes) “across a range of study participation [and use] thresholds” [1], e.g., N exposed, N consented, N used more than x times, N used more than y weeks, N participants “used” the intervention/comparator at speci¦c pre-de¦ned time points of interest (in absolute and relative numbers per group). Always clearly de¦ne “use” of the intervention. 16) For each group, number of participants (denominator) included in each analysis and whether the analysis was by original assigned groups 16) For each group, number of participants (denominator) included in each analysis and whether the analysis was by original assigned groups 16-i) Report multiple “denominators” and provide de¦nitions Report multiple “denominators” and provide de¦nitions: Report N’s (and effect sizes) “across a range of study participation [and use] thresholds” [1], e.g., N exposed, N consented, N used more than x times, N used more than y weeks, N participants “used” the intervention/comparator at speci¦c pre-de¦ned time points of interest (in absolute and relative numbers per group). Always clearly de¦ne “use” of the intervention. 16-ii) Primary analysis should be intent-to-treat Primary analysis should be intent-to-treat, secondary analyses could include comparing only “users”, with the appropriate caveats that this is no longer a randomized sample (see 18-i). 16-ii) Primary analysis should be intent-to-treat Primary analysis should be intent-to-treat, secondary analyses could include comparing only “users”, with the appropriate caveats that this is no longer a randomized sample (see 18-i). ) y y Primary analysis should be intent-to-treat, secondary analyses could include comparing only “users”, with the appropriate caveats that this is no longer a randomized sample (see 18-i). subitem not at all important 1 2 3 4 5 essential subitem not at all important 1 2 3 4 5 essential subitem not at all important Does your paper address subitem 16-i? * Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or brie§y explain why the item is not applicable/relevant for your study 214 participants were assigned to the iCBT treatment group (107) and waiting- list (WL) control group (107). The effect of treatment on PHQ-9 and GAD-7 severity scores was evaluated separately for each measure using repeated- measures linear mixed-effects models ¦t with restricted maximum likelihood in the R package lme4. For each measure, total scores were modelled using ¦xed effects of time, treatment group and the interaction between time and treatment group and a random effect of individual/id. The signi¦cance of each ¦xed effect was evaluated with t-tests using Satterthwaite’s degrees of freedom method. Does your paper address subitem 16-ii? Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or brie§y explain why the item is not applicable/relevant for your study Your answer Your answer 17a) For each primary and secondary outcome, results for each group, and the estimated effect size and its precision (such as 95% con¦dence interval) https://docs.google.com/forms/d/e/1FAIpQLSfZBSUp1bwOc_OimqcS64RdfIAFvmrTSkZQL2-3O8O9hrL5Sw/viewform? m/forms/d/e/1FAIpQLSfZBSUp1bwOc_OimqcS64RdfIAFvmrTSkZQL2-3O8O9hrL5Sw/viewform?hl=en_US&formkey=dG 7/17/2019 CONSORT-EHEALTH (V 1.6.1) - Submission/Publication Form Does your paper address CONSORT subitem 17a? * Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or brie§y explain why the item is not applicable/relevant for your study PHQ-9: The iCBT Group and WL control group samples were compared using linear mixed models (LMM). LMM including ¦xed effects for time showed signi¦cant effects post-treatment (t=-5.079, df=38.23, p=<.001) within the iCBT treatment group. The effects were also maintained at 3-months follow up (t=-4.668, df=39.62, p=<.001) for the treatment group. The results within groups for the treatment group yielded a large effect size post-treatment d=1.44, p= <.001) and this was maintained at 3-month follow-up (d=1.81, p=<.001). Similarly, results showed a large effect size between groups (d=.91, p=<.001). GAD-7: Similarly, the iCBT group and WL control group samples were compared using LMM. Within the iCBT group, time-point was a signi¦cant predictor of GAD- 7 score with a signi¦cant effect from baseline to post-treatment (t=-2.632, df=37.83, p=<.012) and at 3-months follow-up (t=-2486, df=38.8, p=<.017) within the treatment group. The results within groups for the treatment group yielded a medium-large effect size post-treatment (d=0.73 p=<.001) and this was maintained at 3-month follow-up (d=0.52, p=<.001). The mean scores indicated that the treatment group reported signi¦cantly lower levels of anxiety symptoms post-treatment than those in the WL control group, yielding a medium post- treatment effect size between the groups for the intervention (d=.60, p=<.001). PHQ-9: The iCBT Group and WL control group samples were compared using linear mixed models (LMM). LMM including ¦xed effects for time showed signi¦cant effects post-treatment (t=-5.079, df=38.23, p=<.001) within the iCBT treatment group. The effects were also maintained at 3-months follow up (t=-4.668, df=39.62, p=<.001) for the treatment group. The results within groups for the treatment group yielded a large effect size post-treatment d=1.44, p= <.001) and this was maintained at 3-month follow-up (d=1.81, p=<.001). N/A 18) Results of any other analyses performed, including subgroup analyses and adjusted analyses, distinguishing pre- speci¦ed from exploratory 17a-i) Presentation of process outcomes such as metrics of use and intensity of use In addition to primary/secondary (clinical) outcomes, the presentation of process outcomes such as metrics of use and intensity of use (dose, exposure) and their operational de¦nitions is critical. This does not only refer to metrics of attrition (13-b) (often a binary variable), but also to more continuous exposure metrics such as “average session length”. These must be accompanied by a technical description how a metric like a “session” is de¦ned (e.g., timeout after idle time) [1] (report under item 6a). subitem not at all important 1 2 3 4 5 essential https://docs.google.com/forms/d/e/1FAIpQLSfZBSUp1bwOc_OimqcS64RdfIAFvmrTSkZQL2-3O8O9hrL5Sw/viewform?hl=en_US&formkey=dG… 38/47 m/forms/d/e/1FAIpQLSfZBSUp1bwOc_OimqcS64RdfIAFvmrTSkZQL2-3O8O9hrL5Sw/viewform?hl=en_US&formkey=dG https://docs.google.com/forms/d/e/1FAIpQLSfZBSUp1bwOc_OimqcS64RdfIAFvmrTSkZQL2-3O8O9hrL5Sw/viewform? 7/17/2019 CONSORT-EHEALTH (V 1.6.1) - Submission/Publication Form Does your paper address subitem 17a-i? Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or brie§y explain why the item is not applicable/relevant for your study Your answer Your answer 17b) For binary outcomes, presentation of both absolute and relative effect sizes is recommended Does your paper address CONSORT subitem 18? * Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or brie§y explain why the item is not applicable/relevant for your study Satisfaction with treatment (SAT): After 7 weeks, the participants (n=40); also completed the SAT questionnaire. Most of the users were happy to use a computer to access their treatment (76%, n= 30); and found the interventions easy to use (82%, n=32). Furthermore, the majority of participants (68%, n=27), found the online treatment helpful Does your paper address CONSORT subitem 17b? * Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or brie§y explain why the item is not applicable/relevant for your study 18-i) Subgroup analysis of comparing only users ) g p y p g y A subgroup analysis of comparing only users is not uncommon in ehealth trials, but if done, it must be stressed that this is a self-selected sample and no longer an unbiased sample from a randomized trial (see 16-iii). https://docs.google.com/forms/d/e/1FAIpQLSfZBSUp1bwOc_OimqcS64RdfIAFvmrTSkZQL2-3O8O9hrL5Sw/viewform?hl=en_US&formkey=dG… 39/47 7/17/2019 CONSORT-EHEALTH (V 1.6.1) - Submission/Publication Form 19) All important harms or unintended effects in each group (for speci¦c guidance see CONSORT for harms) (for speci¦c guidance see CONSORT for harms) Does your paper address subitem 18-i? Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or brie§y explain why the item is not applicable/relevant for your study Your answer Your answer 19-i) Include privacy breaches, technical problems 19-i) Include privacy breaches, technical problems Include privacy breaches, technical problems. This does not only include physical “harm” to participants, but also incidents such as perceived or real privacy breaches [1], technical problems, and other unexpected/unintended incidents. “Unintended effects” also includes unintended positive effects [2]. Does your paper address CONSORT subitem 19? * Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or brie§y explain why the item is not applicable/relevant for your study The study did not involve any physical o social harm to participants. However, there may be some indirect risk of harm given that the online intervention promotes looking after one’s body through exercising and healthy lifestyle. We did not ¦nd any unintended effects in each group before, during and at the end of the treatment. Does your paper address subitem 19-i? Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or brie§y explain why the item is not applicable/relevant for your study https://docs.google.com/forms/d/e/1FAIpQLSfZBSUp1bwOc_OimqcS64RdfIAFvmrTSkZQL2-3O8O9hrL5Sw/viewform?hl=en_US&formkey=dG… 40/47 m/forms/d/e/1FAIpQLSfZBSUp1bwOc_OimqcS64RdfIAFvmrTSkZQL2-3O8O9hrL5Sw/viewform?hl=en_US&formkey=dG https://docs.google.com/forms/d/e/1FAIpQLSfZBSUp1bwOc_OimqcS64RdfIAFvmrTSkZQL2-3O8O9hrL5Sw/viewform? 7/17/2019 CONSORT-EHEALTH (V 1.6.1) - Submission/Publication Form 19-ii) Include qualitative feedback from participants or observations from staff/researchers Include qualitative feedback from participants or observations from staff/researchers, if available, on strengths and shortcomings of the application, especially if they point to unintended/unexpected effects or uses. This includes (if available) reasons for why people did or did not use the application as intended by the developers. subitem not at all important 1 2 3 4 5 essential subitem not at all important 1 2 3 4 5 essential Does your paper address subitem 19-ii? Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or brie§y explain why the item is not applicable/relevant for your study Your answer 22) Interpretation consistent with results, balancing bene¦ts and harms, and considering other relevant evidence NPT: In addition, take into account the choice of the comparator, lack of or partial blinding, and unequal expertise of care providers or centers in each group https://docs.google.com/forms/d/e/1FAIpQLSfZBSUp1bwOc_OimqcS64RdfIAFvmrTSkZQL2-3O8O9hrL5Sw/viewform?hl=en_US&formkey=dG… 41/47 m/forms/d/e/1FAIpQLSfZBSUp1bwOc_OimqcS64RdfIAFvmrTSkZQL2-3O8O9hrL5Sw/viewform?hl=en_US&formkey=dG https://docs.google.com/forms/d/e/1FAIpQLSfZBSUp1bwOc_OimqcS64RdfIAFvmrTSkZQL2-3O8O9hrL5Sw/viewform? 7/17/2019 CONSORT-EHEALTH (V 1.6.1) - Submission/Publication Form 22-ii) Highlight unanswered new questions, suggest future research Highlight unanswered new questions, suggest future research. Highlight unanswered new questions, suggest future research. Does your paper address subitem 22-i? * Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or brie§y explain why the item is not applicable/relevant for your study The discussion sumarise primary outcomes and process. "The results show that the treatment group demonstrated statistically signi¦cant decreases in depressive symptoms from baseline to post-treatment, relative to pre-post change in the waiting list group, and this reduction in symptoms was maintained at a 3-month follow up. Furthermore, a signicantly larger proportion of the iCBT group achieved reliable change, compared to the waiting list control group. The results from the present study, thus, showed that the culturally adapted cognitive-behavioural internet-delivered programme, Space from Depression is effective in reducing depressive symptoms in comparison to a waiting list control group". Does your paper address subitem 22-ii? Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or brie§y explain why the item is not applicable/relevant for your study Your answer Your answer 20) Trial limitations, addressing sources of potential bias, imprecision, and, if relevant, multiplicity of analyses https://docs.google.com/forms/d/e/1FAIpQLSfZBSUp1bwOc_OimqcS64RdfIAFvmrTSkZQL2-3O8O9hrL5Sw/viewform? m/forms/d/e/1FAIpQLSfZBSUp1bwOc_OimqcS64RdfIAFvmrTSkZQL2-3O8O9hrL5Sw/viewform?hl=en_US&formkey=dG 7/17/2019 CONSORT-EHEALTH (V 1.6.1) - Submission/Publication Form Does your paper address subitem 20-i? * Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or brie§y explain why the item is not applicable/relevant for your study The study included the trial limitations. For instance, "A noted limitation is that the study did not include an o¨cial depression diagnosis of participants". "Due to the high attrition of research data it would not have made sense to complete a true ITT analysis including appropriate imputation of missing values". "The research attrition was high at post-treatment and it was even higher at follow- up". 21) Generalisability (external validity, applicability) of the trial ¦ndings NPT: External validity of the trial ¦ndings according to the intervention, comparators, patients, and care providers or centers involved in the trial 20-i) Typical limitations in ehealth trials Typical limitations in ehealth trials: Participants in ehealth trials are rarely blinded. Ehealth trials often look at a multiplicity of outcomes, increasing risk for a Type I error. Discuss biases due to non-use of the intervention/usability issues, biases through informed consent procedures, unexpected events. subitem not at all important 1 2 3 4 5 essential 21-i) Generalizability to other populations Generalizability to other populations: In particular, discuss generalizability to a general Internet population, outside of a RCT setting, and general patient population, including applicability of the study results for other organizations subitem not at all important 1 2 3 4 5 essential subitem not at all important 1 2 3 4 5 essential 9 CONSORT-EHEALTH (V 1.6.1) - Submission/Publication Form subitem not at all important 1 2 3 4 5 essential OTHER INFORMATION 23) Registration number and name of trial registry 24) Where the full trial protocol can be accessed, if available 21-ii) Discuss if there were elements in the RCT that would be different in a routine application setting Discuss if there were elements in the RCT that would be different in a routine application setting (e.g., prompts/reminders, more human involvement, training sessions or other co-interventions) and what impact the omission of these elements could have on use, adoption, or outcomes if the intervention is applied outside of a RCT setting. Does your paper address subitem 21-ii? Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or brie§y explain why the item is not applicable/relevant for your study Your answer Does your paper address CONSORT subitem 23? * Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or brie§y explain why the item is not applicable/relevant for your study Trial registration number: Clinical trials NCT03062215 7/17/2019 CONSORT-EHEALTH (V 1.6.1) - Submission/Publication Form 21-ii) Discuss if there were elements in the RCT that would be different in a routine application setting 21-ii) Discuss if there were elements in the RCT that would be different in a routine application setting OTHER INFORMATION 23) Registration number and name of trial registry Does your paper address subitem 21-ii? Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or brie§y explain why the item is not applicable/relevant for your study Your answer 21-ii) Discuss if there were elements in the RCT that would be different in a routine application setting Discuss if there were elements in the RCT that would be different in a routine application setting (e.g., prompts/reminders, more human involvement, training sessions or other co-interventions) and what impact the omission of these elements could have on use, adoption, or outcomes if the intervention is applied outside of a RCT setting. subitem not at all important 1 2 3 4 5 essential subitem not at all important 1 2 3 4 5 essential subitem not at all important 1 2 3 4 5 essential Does your paper address subitem 21-i? Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or brie§y explain why the item is not applicable/relevant for your study Your answer https://docs.google.com/forms/d/e/1FAIpQLSfZBSUp1bwOc_OimqcS64RdfIAFvmrTSkZQL2-3O8O9hrL5Sw/viewform? Does your paper address CONSORT subitem 23? * Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or brie§y explain why the item is not applicable/relevant for your study Trial registration number: Clinical trials NCT03062215 24) Where the full trial protocol can be accessed, if available m/forms/d/e/1FAIpQLSfZBSUp1bwOc_OimqcS64RdfIAFvmrTSkZQL2-3O8O9hrL5Sw/viewform?hl=en_US&formkey=dG https://docs.google.com/forms/d/e/1FAIpQLSfZBSUp1bwOc_OimqcS64RdfIAFvmrTSkZQL2-3O8O9hrL5Sw/viewform? 7/17/2019 CONSORT-EHEALTH (V 1.6.1) - Submission/Publication Form Does your paper address CONSORT subitem 24? * Cite a Multimedia Appendix, other reference, or copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or brie§y explain why the item is not applicable/relevant for your study The published trial protocol was cited on the manuscript. Salamanca-Sanabria, Richards, Timulak, L., Castro-Camacho, L., Mojica-Perilla, M., & Parra-Villa, Y. (2018). Assessing the e¨cacy of a culturally adapted cognitive behavioural internet-delivered treatment for depression: protocol for a randomised controlled trial. BMC psychiatry, 18(1), 53. Also, readers may access to the programme asking for a demo in www.silvercloudhealth.com or the principal and secondary author from this paper. 25) Sources of funding and other support (such as supply of drugs), role of funders 25) Sources of funding and other support (such as supply of drugs), role of funders Does your paper address CONSORT subitem 25? * Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or brie§y explain why the item is not applicable/relevant for your study The study was funding by Irish Research Council (IRC) and SilverCloud Health in Dublin, Ireland. The principal researcher is the recipient of an Irish Research Council (IRC) scholarship award [EPSPG/2014/98] under the Enterprise/ Academia collaboration scheme with SilverCloud Health. X27-i) State the relation of the study team towards the system being evaluated In addition to the usual declaration of interests (¦nancial or otherwise), also state the relation of the study team towards the system being evaluated, i.e., state if the authors/evaluators are distinct from or identical with the developers/sponsors of the intervention. subitem not at all important 1 2 3 4 5 essential m/forms/d/e/1FAIpQLSfZBSUp1bwOc_OimqcS64RdfIAFvmrTSkZQL2-3O8O9hrL5Sw/viewform?hl=en_US&formkey=dG https://docs.google.com/forms/d/e/1FAIpQLSfZBSUp1bwOc_OimqcS64RdfIAFvmrTSkZQL2-3O8O9hrL5Sw/viewform? 7/17/2019 CONSORT-EHEALTH (V 1.6.1) - Submission/Publication Form Any other comments or questions on CONSORT EHEALTH Your answer STOP - Save this form as PDF before you click submit To generate a record that you ¦lled in this form, we recommend to generate a PDF of this page (on a Mac, simply select "print" and then select "print as PDF") before you submit it. When you submit your (revised) paper to JMIR, please upload the PDF as supplementary ¦ When you submit your (revised) paper to JMIR, please upload the PDF as supplementary ¦le. Don't worry if some text in the textboxes is cut off, as we still have the complete information in our database Thank you! Don't worry if some text in the textboxes is cut off, as we still have the complete information in our database. Thank you! Final step: Click submit ! Click submit so we have your answers in our database! Never submit passwords through Google Forms. This form was created outside of your domain. Report Abuse - Terms of Service Forms Would you like to become involved in the CONSORT EHEALTH group? Would you like to become involved in the CONSORT EHEALTH group? This would involve for example becoming involved in participating in a workshop and writing an "Explanation and Elaboration" document group? This would involve for example becoming involved in participating in a workshop and writing an "Explanation and Elaboration" document g p This would involve for example becoming involved in participating in a workshop and writing an "Explanation and Elaboration" document Does your paper address subitem X27-i? Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or brie§y explain why the item is not applicable/relevant for your study Your answer Your answer About the CONSORT EHEALTH checklist yes, major changes yes, minor changes no Your answer The checklist implicates reviewing the manuscript several times and to be sure that each item was ¦ne. Spending about 3 days, working 5 hours on it. As a result of using this checklist, do you think your manuscript has improved? * https://docs.google.com/forms/d/e/1FAIpQLSfZBSUp1bwOc_OimqcS64RdfIAFvmrTSkZQL2-3O8O9hrL5Sw/viewform? m/forms/d/e/1FAIpQLSfZBSUp1bwOc_OimqcS64RdfIAFvmrTSkZQL2-3O8O9hrL5Sw/viewform?hl=en_US&formkey=dG CONSORT-EHEALTH (V 1.6.1) - Submission/Publication Form 7/17/2019 Forms https://docs.google.com/forms/d/e/1FAIpQLSfZBSUp1bwOc_OimqcS64RdfIAFvmrTSkZQL2-3O8O9hrL5Sw/viewform?hl=en_US&formkey=dG… 47/47 1FAIpQLSfZBSUp1bwOc_OimqcS64RdfIAFvmrTSkZQL2-3O8O9hrL5Sw/viewform?hl=en_US&formkey=dG… 47/47 https://docs.google.com/forms/d/e/1FAIpQLSfZBSUp1bwOc_OimqcS64RdfIAFvmrTSkZQL2-3O8O9hrL5Sw/viewform?
https://openalex.org/W3141345147	https://www.researchsquare.com/article/rs-351612/latest.pdf	English	null	Card Sorting: Challenges in Mathematics during Emergencies and Crises	Research Square (Research Square)	2,021	cc-by	12,779	Card Sorting: A new pedagogy for understanding challenges in Mathematics during Emergencies and Crises Sione Paea (  sione.paea@usp.ac.fj ) The University of the South Pacific Bibhya Sharma The University of the South Pacific Gabiriele Bulivou The University of the South Pacific Mele Katea Paea The University of the South Pacific The University of the South Pacific Research Article Keywords: Card sorting, Challenges, Similarity matrix, 3D Cluster View (3DCV), Higher Education Institutions, Mathematics Posted Date: May 4th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-351612/v2 DOI: https://doi.org/10.21203/rs.3.rs-351612/v2 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/31 Page 1/31 Abstract Interest in challenges faced by university students during COVID-19 has led to research and development initiatives that include educational, technological, economical and socio-cultural provisions. Despite these initiatives, little is known about the usage of open card sorting, similarity matrix, and Hierarchical Clustering Method - 3D Cluster View algorithm in understanding and analysing mathematics challenges in a regional university during emergencies and crisis. This paper presents findings from a study that explored the challenges encountered by first-year mathematics students in a South Pacific institution. The findings reveal seven challenges: i) financial hardship; ii) motivational challenge; iii) moodle issues; iv) lack of face to face interactions; v) problem with course delivery; vi) internet challenge; and vi) home disturbances. A heptagon model is presented with possible solutions for the challenges identified by participants. The findings point to the complex inter-relationship between the institution’s emergency remote teaching, students’ learning needs, and students’ dynamic socio-cultural environments as important factors for delivering quality mathematics learning during a pandemic. This paper highlights the contribution of card sorting, as a new pedagogy, to the field of educational research as a provider of new learning analytics for desirable learning outcomes in a given pandemic. Decisionmakers and Policymakers of Higher Education Institutions around the world may benefit from these findings while formulating strategies to support first-year mathematics students during the current and future pandemics. 1 Introduction The world is currently experiencing and gradually responding to a widespread of transmissible respiratory disease caused by an original coronavirus named COVID-19. The disease was declared a global pandemic by the World Health Organisation (WHO) on March 11, 2020. Over 1.5 billion students in 165 countries are affected by COVID-19 related school closures, equivalent to 87% of the world’s student population. To protect this population from the spread of COVID-19 as a contagious and deadly virus, the emergency remote teaching (ERT) has been an overwhelming response of many Higher Education Institutions (HEI) around the world (Mulenga and Marbán 2020). The global shift to ERT (Bozkurt and Sharma 2020; Hodges et al. 2020; Karakaya 2021; Trust and Whalen 2020; West et al. 2020) and its actual application using online platforms has posed many unexpected challenges that students, parents, and institutions were not adequately prepared for. This includes issues regarding technology, home learning environment, internet network capacity, and time conflicts between work and online learning sessions (Mohmmed et al. 2020). In most cases, students have complained about difficulties in gaining access to educational resources and completing activities and assessments. On the other hand, the universities are concerned with the lack of student engagement in an ERT learning environment (Affouneh 2020). Page 2/31 The challenges of supporting learner interests and understanding through online education are well- documented, which include feeling of disconnection (Choudhury and Pattnaik 2020), difficulty to meet individual learning needs (Haavind and Sistek-Chandler 2015) and lack of self-direction (Beaven et al. 2014). These challenges were exacerbated during the pandemic, where many educators and learners suddenly immersed in unfamiliar and unaffordable situations (Blum-Smith et al. 2020). Studies on understanding Page 2/31 Page 2/31 university teachers’ design work emphasise the significance of looking into what teachers actually do in the process of instructional designing and planning activities (Bennet et al., 2017) because learning design during an ERT can be dynamically shaped (Karakaya, K., 2020). The fact that learning mathematics in the online environment has always been an issue, sudden shifts to online in emergencies without due process will make learning even more difficult and unsustainable. This underscores the significance of the current study and the crucial role of the university in transforming teaching-learning practices towards a sustainable learning environment. Research explores the challenges faced by first-year mathematics students during the unexpected shift to ERT due to COVID-19 lockdown restrictions in a regional university. 1 Introduction The objective is to have a better understanding of those challenges and to propose a new learning support model that can strengthen quality learning in mathematics in an emergency. Such a model can guide the institution to cultivate stronger learning strategies of resilience during a pandemic and to ensure better preparation for the next inevitable emergency and crisis. The term challenge is defined in this paper as the difficulties, problems or issues that affect students’ learning ability to achieve during the sudden change of learning modes in an emergency response. An open card sorting (OCS) (Paea et al. 2020) was employed to explore the topic by drawing cards initially from the institution’s understanding of students’ experiences whilst studying mathematics during the COVID- 19 lockdown. A total of seven challenges were commonly shared amongst participants’ findings: i) financial hardship; ii) motivational challenge; iii) moodle issue; iv) lack of face-to-face (f2f) interaction; v) problem with course delivery; vi) internet challenge; and vii) home disturbance. These challenges are made up of different inter-related components that should be understood and addressed from a dynamic multi-dimensional perspective. It highlights the complex way of life that Pacific students live their lives in a given context, which cannot be separated from their learning experience during emergencies and crises. This provides direction to the university about the need for a long-term emergency system in place that would involve clear investment and coordination for the future. The main contributions of this paper are as follows: 1. the important role of card sorting in the field of educational research as a provider of new learning analytics for quality learning in mathematics and desirable learning outcomes from a range of known and unknown inputs in a given pandemic; Card sorting has classified and analysed the findings in a more organised manner; 2. the appropriate use of fit values of stress and R-squared to choose the best fit model data. New insights suggest the three-dimensional solution an appropriate model for card sorting datasets; 2. the appropriate use of fit values of stress and R-squared to choose the best fit model data. New insights suggest the three-dimensional solution an appropriate model for card sorting datasets; 3. the use of card sorting and resulting analytics points to a significant association between emergency remote learning in mathematics, socio-cultural student challenges, and quality learning in a given pandemic. 1 Introduction The implication is that the 3DCV process was effective in determining relationships among challenges (items) in a manner that is more effective than applying a general logic approach; 4. new direction beyond student perceptions and outcome measures. The heptagon model can replicate with a broader coverage of the major conditions that contribute to online instruction quality at times of 4. new direction beyond student perceptions and outcome measures. The heptagon model can replicate with a broader coverage of the major conditions that contribute to online instruction quality at times of Page 3/31 Page 3/31 Page 3/31 emergencies and crises; emergencies and crises; 5. use of new heptagon model for better comprehending of the complex nature of mathematics students’ learning experiences in a South Pacific regional university, calling for a more inclusive, affordable and sustainable ERT. The findings can be applied in any learning environment with special attention given to learners’ dynamic socio-cultural backgrounds that should include contextualisation. The paper consists of 8 sections, including this introductory section. The next two sections provide a brief description of the research context and the literature relevant to the research topic. This is followed by research strategies in section four then the questionnaire in section five. Later in section six is the presentation and discussion of findings followed by demonstrating potential solutions for participants’ challenges in a heptagon model in section seven. The final section concludes the paper. 2 Research Context The USP is one of only two regional universities in the world. It is jointly owned by the governments of 12 member countries across the South Pacific region: Cook Islands, Fiji, Kiribati, Marshall Islands, Nauru, Niue, Samoa, Solomon Islands, Tokelau, Tonga, Tuvalu, and Vanuatu. The role of the USP in developing the knowledge economy of the region places the university in a unique position in relation to education and research. The university has campuses in all member countries, and the current study was fully implemented at the main campus, Laucala, in Fiji. After the announcement of the first case of COVID-19 in Fiji on March 19, the USP was totally closed down for 2 weeks, March 26 – April 9, before the University began its emergency remote classes in the second half of the first semester for 7 weeks from April to June 2020. The courses returned to normal blended mode (f2f and online) at the beginning of the second semester of 2020. During the 7 weeks of ERT at the USP, the authors found student’s self-discipline at the core of their own success. Mathematics students with high self-discipline could follow instructions and complete work on time, whereas those with low self-discipline would be impossible. When students’ learning behaviours cannot be monitored remotely, their lack of self-discipline would greatly impact their learning outcomes. The unexpected shift to ERT also required teaching staff to use new software and unfamiliar online tools without having proper technical support. A significant challenge for HEI teachers has been their lack of the pedagogical content knowledge (PCK) (Tondeur et al. 2020; Shulman 1987) needed for teaching online (Kali, Goodyear and Markauskaite 2011; Ching et al. 2018). Accordingly, the use of digital and technological resources for ERT of mathematics has increased sharply. Still, its success can only be guaranteed when the teachers are competent and ready to develop and utilise these resources. This reinforces the need for HEI to be more prepared for the future, emphasising the important contribution the current study can offer to achieve that from a Pacific learning perspective during an emergency. 3 Literature Review Some recent studies on ERT highlight different types of challenges experienced by students during the unexpected educational shift (e.g., (Aguliera and Nightingale-Lee 2020; Bozkurt and Sharma 2020; Hodges et Page 4/31 Page 4/31 al. 2020; Mohmmed et al. 2020; Mulenga & Marbán 2020). A study that explored students' lived experiences as impacted by emergency shift to remote teaching in the United States of America confirmed that students with existing educational inequality had been exposed to more learning inequalities during the abrupt shift to ERT (Aguliera and Nightingale-Lee 2020; Trust and Whalen 2020). Students had to provide their own learning resources that put them at a great disadvantage in relation to the unsafe learning environment, poor access to the internet and inability to possess electronic devices (Aguliera and Nightingale-Lee 2020; Zilka et al. 2018). Those students who do not have access to laptops or high-speed internet at home would experience more severe learning challenges, which may delay the acceptance of technology-enabled education (Zilka et al. 2018). These resources (non-financial such as computer, internet access) are crucial to obtain HEI goals and become essential home possessions during emergencies and crises The ERT also comprises of issues including time management, technology illiteracy, students’ assessment, communication, and the lack of in-person interaction (e.g., (Karakaya 2021; Steele 2019). Interacting online requires educators to rethink online pedagogy so as to support meaningful (higher-order) learning and its assessment (Gikandi et al. 2011; Ćukušić et al. 2014). Aside from online infrastructure challenges, Bozkurt and Sharma (2020) argue for more attention to the lack of empathetic support for students during the crisis of COVID-19 because “students will remember not the educational content delivered, but how they felt during these hard times” (Bozkurt and Sharma 2020). This presents the importance of motivation and self-esteem protection, during an ERT, for resilience and quality learning. Mulenga and Marbán (2020) explore the perspectives of teachers who were engaged in teaching mathematics online during COVID-19 and found that educators, teachers, and ERT staff need better training and support for using online tools (Trust and Whalen 2020). These challenges give direction to the future, calling for the institution to work in collaboration with stakeholders to offer better solutions in preparation for future interruptions (Bozkurt and Sharma 2020) and to formulate more forward-looking strategies towards improving teaching-learning activities during COVID-19 ERT (Mohmmed et al. 2020). 3 Literature Review With the rise in the use of online modalities during COVID-19, it is necessary to assess their effectiveness regarding teaching and learning from different stakeholders (Schwartz et al. 2020). The nature of online learning means that working in partnership with numerous digital innovators and instructors who see technology as a method of solving problems and reaching new learners is needed. The use of card sorting to explore mathematics students’ challenges during a pandemic in the South Pacific has been absent. However, the practical guidance on card sorting in a Pacific HEI will help to understand how it can be implemented appropriately in the Pacific context (Paea et al. 2020). Card Sorting is utilised to evaluate the information architecture (IA) of a finding and to design a navigation structure that can offer an exciting variety of content and functionality (Righi et al. 2013). The IA of the current research refers to the challenges that emerged from this study in relation to first-year mathematics students’ learning experiences during the emergency and crisis of COVID-19. It provides insight into users’ mental models (Katsanos et al., 2019), revealing how they often implicitly group, sort, and label tasks and content within their own heads according to their understanding. The term card sort applies to a wide range of activities, including ordering, grouping, and/or naming objects or concepts (Paea and Baird 2018). With card sorting, researchers learn how users categorise and label their thoughts to express their feeling and experience in a challenging Page 5/31 Page 5/31 situation. Card sorting provides a map of users’ knowledge of the content and where they might look for the content within the design of an actual interface display. situation. Card sorting provides a map of users’ knowledge of the content and where they might look for the content within the design of an actual interface display. Card sorting also has applications in visualisation research. For example, it has been used to explore people’s mental models of classifying visualisation methods. It has also been used as a way to conduct a task analysis of geovisualisation tools and interactions (Lloyd et al. 2007). Using the card sorting method in this study aims to target the users' understanding of the challenges based on their experiences during the shift from f2f to ERT. This is also the first time to utilise the card sorting method to generate useful insights about the research question. 3 Literature Review Efforts to develop, understand, and productively use card sorting data have organised into a field of educational research. The need for a better understanding of first-year mathematics students’ challenges during COVID-19 using card sorting approach in a Pacific learning context is highlighted by its absence in the literature. It appears from the ERT literature that students’ challenges during the emergency and crisis of a pandemic is complex in a way it is based on the dynamic inter-relationships between students’ home learning environments, students-teachers digital literacy, course delivery, internet network access, students’ motivation, and the socio- cultural contexts in which students live and operate. These challenges set the foundation for exploring the research topic, which offers timely insights to the gap in the literature of interconnectivity between card sorting, quality learning in mathematics, ERT, and Pacific regional HEI. 4.1 Population, setting, and sample The target population is the first-year or 100 level students who studied mathematics at the USP in semester one (February – June) of 2020. Targeting first year f2f mathematics students is essential for understanding their learning needs as new entrants to the university during a pandemic and how best to support them towards persistence or successful completion of mathematics courses. This also provides a more realistic insight into the challenges that mathematics students faced while going through unprecedented change to teaching and learning during the university’s COVID-19 lockdown. Since it was impractical to explore the topic with the complete USP population across the university’s regional campuses, the research team chose the Laucala campus in Suva, Fiji, because this setting recruits the highest proportion of first-year face-to-face mathematics students. It is also the most central setting considering COVID-19 restrictions on regional travels in the South Pacific. The respondents in the target populations were recruited by convenience sampling facilitated by advertising on MA111 moodle page. The study recruited a total of 32 (16 men and 16 women) first-year mathematics students who are currently studying at the USP and citizens of the university’s country members. Such a recruitment reflects the diverse realities shaping the formation of learning at the USP, and the complexities involved with direct and indirect impacts of pandemic on students’ learning experiences. The participants’ ages ranged from 18 to 28 (M = 20 and SD = 2.9). Page 6/31 Page 6/31 The authors’ decision to recruit 32 participants is based on their experience as card sorting researchers and from reviewing of previous card sorting studies that have examined the adequate sample size needed to produce high-quality representation (Optimal Workshop; Tullis and Wood 2004; Wood and Wood 2008). Tullis and Wood (2004) found that a sample size of 20-30 participants explains 90-95% of the true information space structure, with diminishing returns in explanatory power as the sample size increases beyond 30. Wood and Wood (2008) confirmed that recruiting as few as 25-30 participants would likely yield results similar to those of several hundred provided these participants are representative of actual users and are familiar with the domain being considered. Rosas and Kane (2012) indicated the variability in each study's stress values was dramatic when about 15 or fewer participants were included. As the number of participants for each of their five studies reached about 35 participants, substantial improvements in stress (i.e. 4.1 Population, setting, and sample lower stress values) were observed. However, beyond 40 participants, only marginal improvements in stress were detected. These findings suggest between 20 and 30 participants is warranted to maximise the consistency of fit in the concept mapping representation by minimising the variability in the stress value found with smaller groups of participants. For open card sorting, the ideal sample size is between 30 and 50 participants as supported by Card sorting 101 (Optimal Workishop). Completed card sorts within this sample interval would be easy to identify ideas and consensus (Optimal Workshop). Also, keep in mind that the more participants you have completed your card sort, the potential for more complexity in your analysis increases as well. This is simply because narrowing down the most effective structure from 40 different suggested categorisations will probably be easier than 200 various suggestions. Also, Research suggests a minimum of 15 users to obtain robust data from open card sorts (Katsanos 2018; Nielson 2004; Tullis and Wood 2005), thus our studies had adequate sample size. Hence the authors decided to recruit 32 participants as the sample size. Participants were recruited through a variety of means including personal contacts, referrals and voluntary. The authors then administered the research announcement to the students who were enrolled in MA111. The Moodle message, course announcement via Moodle and email distribution were used to inform the students about the research and encourage them to participate voluntarily. Participants were also recruited using an informal snowball process that was based on researchers’ cultural knowledge and skills of recruiting Pacific participants through networking and relationship building (Paea et al. 2020). This type of recruitment is important for building trust and respect amongst participants and the researcher because Pacific people can willingly partake when they trust the researcher; and know their contribution is recognised and valued (Paea et al. 2020). Prior to the day of the actual card sorting, a card sorting demonstration video and an information sheet were sent to participants beforehand. This is to provide participants with relevant information about the research objectives, how to do card sorting, and how it would affect them during and after the fieldwork. 4.3 Face-to-face open card sorting The study was implemented through open card sorting (OCS) in f2f mode because the nature of sharing responsibilities and co-constructing meaning between participants and the researchers have strengthened the quality of their relationship and the findings (Paea et al. 2020). On the day of f2f card sorting, one of the researchers welcomed participants by acknowledging their presence, time, and contribution. Participants were allowed to introduce themselves including their Pacific originalities because it gave them a sense of belonging to the card sorting context. The researcher went on to brief participants about the research objectives, informed them that their personal details would be kept confidential, and allowed them to ask questions about the research before they signed the consent form. A pile of 44 physical cards was placed on the table. Participants were asked to sort the cards into groups of similarities and labelled groups according to the challenges they experienced during the unexpected change from f2f to ERL. The participants performed the card sort individually to assure independence of grouping strategies. For each participant, the authors took a photo of the final card sorting and audio-recorded their verbalised thoughts. Additionally, audio recordings were transcribed and analysed to provide valuable insight, detailed data pre-processing and elaborate them to rich visualisation. The participants' details were kept anonymous and the responses were only used for analysis purpose. The actual time of card sorting varied from 30-70 minutes to complete. Some participants created just four categories, while others created more complex classifications involving up to 10 categories (M = 7, SD = 1.5). There were no significant differences between the number of categories formed by males (M = 7) and females (M= 7), t(7) = 0.88, ns, and the number of categories formed was unrelated to age (r = -0.23, ns). Once participants had grouped the challenges, they named each grouping they had formed to help explain commonalities between the challenges contained within the grouping. Figure 1 illustrates participants’ pathway through the f2f OCS during an active card sorting performed by one of the participants. It shows how f2f card sorting is conducted using physical cards in an OCS. During the actual performance, participants were allowed to move cards to ensure their experiences were consolidated. The blue sticky papers on top of each column represent the group numbers with unknown category names, and sorted cards are presented under each blue coloured paper. 4.2 Card size and names A total of 44 physical card names were drawn from the university’s in-house report on the challenges facing first-year mathematics students in the process of a sudden shift to emergency remote learning. The 44 cards Page 7/31 are related to each other in the sense of challenges, but they clearly comprehend the group's own cluster by the participants. This was supported by two mathematics lecturers' experience in the current research team to generate a Pacific solution-based framework to strengthen students’ resilience and retention during a pandemic. Card names represent the challenges, problems, or issues that hinder students’ ability to achieve during the unforeseen shift. The names were made in a lower-level meaning for participants to understand. For instance, the card name ‘internet data is expensive’ means that students cannot access to online learning because of financial hardship and the card names are related to each other. The physical cards are chosen to align with what most studied recommended (Optimal Workshop; Tullis and Wood 2004). are related to each other in the sense of challenges, but they clearly com the participants. This was supported by two mathematics lecturers' exp to generate a Pacific solution-based framework to strengthen students’ pandemic. Card names represent the challenges, problems, or issues tha during the unforeseen shift. The names were made in a lower-level mea For instance, the card name ‘internet data is expensive’ means that stud because of financial hardship and the card names are related to each ot align with what most studied recommended (Optimal Workshop; Tullis a 4.4 Card sorting analysis There were two main phases to the analysis. The first phase was the construction of similarity matrices to test how strongly the group elements of challenges are related to each other. The second phase was a 3DCV analysis of the card-sort data (Optimal Workshop ; Paea & Baird 2018; Paea et al. 2020). To ensure usability and simplicity for better comprehension of participants’ challenges, the category labels were revised. For example, in Group 3 of Table 1 (p 14), there are 3 commonly shared categories based on participants’ data: financial issues (82%), challenges with rolling expenses (80%), and family financial background (78%). Since all these categories represent participants’ financial difficulties, the category name ‘financial hardship’ was used. While these steps can be seen as modifying the original data, it enhances data consistency and clarity without changing the meaning of participants’ original data. This process of co-constructing meaning between participants and the researcher(s) is acceptable in the Pacific Way of carrying out card sorting research (Paea et al. 2020). 4.3 Face-to-face open card sorting The category names were numbered for ease of reference. Page 8/31 Page 8/31 4.4.1 Phase 1: Similarity Matrix A a similarity matrix was constructed to represent the raw card sort data. A matrix was created for every participant, indicating whether each pair of strategies was placed in the same grouping (“1”) or in a different grouping (“0”) in the participant’s card-sort solution. These matrices were then aggregated cell-by-cell to create a matrix with cell values ranging between 0 (if no participants had placed a particular pair of challenges in the same group) and 32 (100%) (if all participants had placed the pair in the same group). The similarity matrix is used to interpret how strongly the group elements of challenges are related to each other. Since the research has considered card names as representation of participants’ learning challenges during an emergency, the similarity matrix in Figure 2 is a straightforward representation of cards combinations. It intends to give insights into the challenges that participants pair together in clusters, which also identify pairs of closely related challenges by assigning them higher similarity than those that are distantly related. The similarity is measured between two individuals in the cards of challenges, with the similarity matrix being formed by combining this information for all pairs of challenges. For instance, the first column of the matrix shows that 87% of participants put the challenges ‘expensive to buy relevant software’ and ‘cannot afford to buy the textbooks’ in the same group; meaning that both cards are interconnected highlighting participants’ financial difficulties as the lead cause of their learning challenges during the pandemic. Looking further down the same column, ‘expensive to buy relevant software’ and ‘not working hard enough on assessment’ were never placed together. This means that participants’ financial and motivational challenges are not related. Accordingly, the strongest pair is positioned at the top left corner, grouping them with the next associated strongest pair that either of those challenges have, and then the process is repeated for that new pair. This way, groups of challenges that are strongly related to each other appear together in the same shade of blue on the similarity matrix. The darker the blue shaded areas where two challenges intersect, the more often they were paired together by the participants (Optimal Workshop; Paea & Baird, 2018; Paea et al., 2020). Page 9/31 Page 9/31 Page 9/31 It can be seen from Figure 2 that the blue shaded areas have an inconsistent pattern. 4.4.1 Phase 1: Similarity Matrix The authors subsequently display the data that are positioned along the right edge in Figure 2 as a line graph (Figure 3) to identify possible major challenges of clusters. The technique can assist in finding a suitable analytical method to analyse the finding. For instance, in Figure 3, the red colour can be potential clusters. The black colour is threatening to decide if these challenges belong to a particular cluster due to low participant agreement. With the inconsistent patterns and low participant agreement, the 3D Cluster View (3DCV) is utilised to visualise the data clearly in Figure 2 by grouping the challenges in 7 clusters, as demonstrated in Figure 5. The next section describes how the authors’ finalised the total number of categories in 7 clusters. It can be seen from Figure 2 that the blue shaded areas have an subsequently display the data that are positioned along the righ identify possible major challenges of clusters. The technique ca method to analyse the finding. For instance, in Figure 3, the red c colour is threatening to decide if these challenges belong to a pa agreement. With the inconsistent patterns and low participant a utilised to visualise the data clearly in Figure 2 by grouping the c Figure 5. The next section describes how the authors’ finalised t Table 1: Total variance explained. 4.4.2 Phase 2: Number of Clusters One important challenge that arises in quantitative analysis of card sort data is deciding the optimal number of clusters. In the initial solution, the number of clusters is equal to the number of cards included in the study, that is 44 physical cards (see Table 1). This paper uses the approached by Katsanoe et al. 2008 based on the widely used eigenvalue-one criterion to identify the optimal number of clusters. Every cluster has an eigenvalue representing the amount of variance accounted for by a given cluster. Usually, the first variables have the greatest eigenvalues. The method identifies the optimal number of clusters in terms of variance explained by implementing an eigenvalue analysis of the challenges’ similarity matrix (Figure 2) and keeping only the eigenvalues greater than 1 (see Table 1). Table 1 shows that only the first seven components have the eigenvalue greater than one. Another method used for factor extraction is the analysis of the scree plot (Cattell 1966) or elbow criterion. According to this criterion, the significant factors are disposed like a cliff, having a big slope while the trivial factors are disposed at the base of the cliff. This is achieved by plotting the eigenvalue against the number of clusters (see Figure 4a). Also, plotting the percentage of variance explained against the number of clusters (see Figure 4b). In Figure 4 a and b we can appreciate that starting with the seven-factor the slope of the curve is relatively small and these factors could be excluded from the model. Nevertheless, Figure 4 is very subjective because the curve's cut-off point is sometimes not very clear. Then we compare the result from Table 1 and figure 4 with the Optimal Workshop 3D Cluster View algorithm. This is calculated simply by taking the average (mean) of the number of categories created by participants in the survey. 3D Cluster View algorithm also provided seven clusters and the result agrees to the finding in Table 1 and Figure 4. Then the author’s concluded the optimal number of clusters is seven. Table 1: Total variance explained. Page 10/31 4.4.3 Phase 3: Stress and Goodness of Fit The goodness of fit of the multidimensional scaling (MDS) results was shown by stress values and squared correlation (r-squared), as displayed in Table 2 and Figure 5. In order to select the best fitting model data, the fit values of stress and R-squared were examined. The stress in three dimensions for the output shown in Figure 5a) is 0.288. MDS literature suggests lower stress values are preferred and reflect better congruence between the raw data and the processed data (Davison 1983; Kruskal 1964). The stress values found in the dataset are typically higher than those recommended in the literature on MDS. Several reasons for the discrepancy have been presented by Trochim (1993) and Kane and Trochim (2007). The r-squared ( ) against dimension is plotted to assist us in choosing the best dimensions. Figure 5b shows that as the number of dimensions increases from three to four, r-square values observed converge and begin to level off. The three dimensions squared correlation (r-squared) value (0.928) approaching 1 (100%) indicates that the MDS Page 11/31 Page 11/31 model can be said to be good (Redell 2019; Seok 2009). This study found that the three-dimensional solution was the appropriate model for the card sorting datasets. Table 2: The numerical fit indexes. Table 2: The numerical fit indexes. Number of dimensions Stress r-squared 1 0.495 0.839 2 0.345 0.903 3 0.288 0.928 4 0.253 0.929 4.4.3 Phase 3: Hierarchical Clustering Method - 3D Cluster View (3DCV) analysis To establish whether the participants’ underlying structure supported the theoretical grouping, we subjected the similarity matrix in Figure 2 to hierarchical cluster analysis. Hierarchical cluster analysis arranges objects (in this case, challenges) into relatively homogeneous groups (Aldenderfer and Blashfield 1984; Antonenko et al. 2012), thus allowing researchers to recognise the commonalities and distinctions relevant to the participant group as a whole. An agglomerative clustering method was selected, whereby the most similar challenges (in this study) were successively merged to produce non-overlapping hierarchically clusters of increasing inclusiveness (Davidson and Ravi 2009; Zhao and Karypis 2005). The average linkage rule (Sokal and Michener 1958), also referred to as the within-group linkage method, was used. This rule joins the two most similar cards together in a cluster and then calculates the average similarity of a card with all other cards within and outside the cluster. A card only joins a cluster if a given level of overall similarity is achieved. The 3DCV is utilised to visualise the data in Figure 2 and Figure 3 more clearly. The main output is a 3D- plotted, which graphically represented how the 44 challenges group into hierarchical clusters. This method reduces the difficulty in interpreting a plot that contains too much data, long labels, and inconsistent patterns. Figure 6 shows seven group of challenges as proposed by 3DCV method from the dataset given in Figure 2, with each cluster shown in different colours. Each point in the visualisation represents a distinct challenge. Challenges that are closer together were more frequently sorted into the same category. Polygons show the group of challenges that are clustered together. Each of these groups can be interpreted as a potential category within an IA. Placing the cursor over any colour ballpoint will highlight the card name that the ballpoint represents. As shown at the bottom right-hand side of Figure 6, the category labels and the challenge names will be highlighted when the cursor is placed over any polygon. To reduce this complex representation to a more simplified and meaningful solution, we also visually inspected it, to determine the Page 12/31 Page 12/31 number of clusters at each polygon shown in Figure 6. We then interpreted the clusters produced at each polygon shown in Table 3. Figure 7 presents a bar graph of Figure 6 to show the number of cards in each cluster for clear visualisation. 5 Questionnaire The second part of the research methodology is an online questionnaire provided to the 32 participants. Since, this is the first study done on the use of card sorting and the students’ challenges during the covid-19, the authors decided to use an online questionnaire to collect the data to identify how the 7 challenges (Figure 5 and 6 and Table 2) were rated by participants. Using online questionnaire methodology enabled the authors to collect information regarding the students' attitude and satisfaction in naming the seven challenges for their learning. The students were given an online questionnaire which was designed using a 7-points in descending order. The question is “Rate the 7 challenges from the highest to least challenges, that is, top challenge = 1 and least challenge = 7. The authors then administered the questionnaire to the participanrs who were participated in the study. The email distribution was used to inform the 32 participants about the questionnaire. The questionnaire was open to students for one week. A student took a maximum time of seven minutes to fill in the questionnaire. The students' responses to the online questionnaire were automatically saved in author’s email. The participant’s responses were confidential. All responses were compiled and analysed as a group. The participants' details were kept anonymous and the responses were only used for analysis purpose. The simplified solution to the participants’ classification is displayed in Table 3. The table also lists the most prototypical challenges from highest level category. 4.4.3 Phase 3: Hierarchical Clustering Method - 3D Cluster View (3DCV) analysis The bar graph reveals the hidden meaning of the challenges in each category. The number of cards indicates how serious the challenges are, meaning that most students encountered a learning challenge during COVID- 19. The higher the number of cards in a cluster, the more frequently the challenge is being faced by students during the COVID-19 crisis. The two category names that contain the highest number of cards are financial issues and students’ motivational challenges. This indicates that the majority of students have considered their ‘financial hardship’ and motivation as major causes of their challenges during the emergency shift. The seven category names are related to each other. In the next section a second data collection method of online questionnaire has been applied to rate the category names. 6 Findings And Discussion The key findings from card sorting analysis are presented in Table 3 with the primary level group number in the first column, group labels in the second column, proposed group label in the third column and the list of card challenges in the final column. For instance, row 1 of Table 3 shows that 82% of participants label primary level Group 1 ‘financial issues’, 80% label it ‘challenges with rolling expenses’, and 78% label it ‘family financial background’. This result suggests that ‘financial hardship’ can be the proposed category label for primary level Group 1 as determined by the list of similar related category labels and similar related card challenges displayed in the third column. A similar application can be repeated for the rest of the proposed group labels in Table 2. Page 13/31 Table 3: The findings from the 3DCV of the dataset in Figure 2. 6 Findings And Discussion Page 14/31 LEVEL 1 GROUP GROUP LABEL PROPOSED GROUP LABEL CARD NAME Group 1 Similar category labels Financial issues (82% ) Challenges with rolling expenses (80%) Family financial background (78%) Financial Hardship 9 Cards No computer, laptop, or mobile device at home for online study Cannot afford to buy the textbook Expensive to buy relevant software Hard to study without a textbook Internet price is expensive No /Limited money to purchase internet data No access to relevant software e.g., Mathematica No bus fare to go to the University campus No electricity at home Group 2 Similar category labels Challenges faced by students (60%) Improper time management (55%) Can make the student fail the course (50%) Motivational challenge 9 Cards Clashes between work and online learning schedules Don’t like online tutorials and online lectures No attendance in tutorial and lab makes students lazy No partial marks for the working during online test Not working hard enough on assessment Poor time & workload management Studying online alone is challenging Time limitation for quizzes and tests cause frustration Too many assessments Group 3 Similar category labels Moodle issues (71% ) Challenges in using moodle (57%) Difficulties in moodles (51%) Moodle Issues 7 Cards Don’t access to solutions & past exam papers on moodle No training on how to do online activities in moodle Not familiar or confuse in using moodle and course shell One satellite session a week is not enough Poor quality of audio and lecture/tutorial videos LEVEL 1 GROUP GROUP LABEL PROPOSED GROUP LABEL CARD NAME Group 1 Similar category labels Financial issues (82% ) Challenges with rolling expenses (80%) Family financial background (78%) Financial Hardship 9 Cards No computer, laptop, or mobile device at home for online study Cannot afford to buy the textbook Expensive to buy relevant software Hard to study without a textbook Internet price is expensive No /Limited money to purchase internet data No access to relevant software e.g., Mathematica No bus fare to go to the University campus No electricity at home Group 2 Similar category labels Challenges faced by students (60%) Improper time management (55%) Can make the student fail the course (50%) Motivational challenge 9 Cards Clashes between work and online learning schedules Don’t like online tutorials and online lectures No attendance in tutorial and lab makes students lazy No partial marks for the working during online test Not working hard enough on assessment Poor time & workload management Studying online alone is challenging Time limitation for quizzes and tests cause frustration Too many assessments Group 3 Similar category labels Moodle issues (71% ) Challenges in using moodle (57%) Difficulties in moodles (51%) Moodle Issues 7 Cards Don’t access to solutions & past exam papers on moodle No training on how to do online activities in moodle Not familiar or confuse in using moodle and course shell One satellite session a week is not enough Poor quality of audio and lecture/tutorial videos PROPOSED GROUP LABEL CARD NAME Page 15/31 Group 2 Similar category labels Challenges faced by students (60%) Improper time management (55%) Can make the student fail the course (50%) Motivational challenge 9 Cards Clashes between work and online learning schedules Don’t like online tutorials and online lectures No attendance in tutorial and lab makes students lazy No partial marks for the working during online test Not working hard enough on assessment Poor time & workload management Studying online alone is challenging Time limitation for quizzes and tests cause frustration Too many assessments Group 3 Similar category labels Moodle issues (71% ) Challenges in using moodle (57%) Difficulties in moodles (51%) Moodle Issues 7 Cards Don’t access to solutions & past exam papers on moodle No training on how to do online activities in moodle Not familiar or confuse in using moodle and course shell One satellite session a week is not enough Poor quality of audio and lecture/tutorial videos Timing of live sessions not aligned to regional campuses outside Fiji Too many information in the course shell can cause confusion Don’t access to solutions & past exam papers on moodle Timing of live sessions not aligned to regional campuses outside Fiji Too many information in the course shell can cause confusion Page 15/31 Group 4 Similar category labels F2f interaction issues (63%) Group work issues (63% ) Lack of face to face components (63%) Lack of f2f interaction 7 Cards Lack of f2f online assessments e.g., online group work Limited opportunity to interact f2f with peers to succeed in the unit Easy to get solutions to quizzes and tests on the internet Lack of student engagement in learning activities No group work incorporated in online learning and assessments No study buddy or partner to study with Struggle to understand math activities and tricky tutorial questions provided online Group 5 Similar category labels Teaching staff Issues (83%) Tutor problem (67% ) Tutor Short fall (67%) Problem with course delivery 6 Cards Typos in lecture notes and examples and solutions having errors cause confusion Lack of mathematics examples from the Pacific context Lack of personal assistance from teaching staff No tutors available weekly for regional students Poor communication and feedback from staff Tutors are less active in the online activities Group 6 Similar category labels Internet challenges (100%) Resources accessibility (80%) Internet issues (67%) Internet challenge 4 Cards A poor internet connection and poor- quality internet Difficulty in downloading large videos file size No access to the computer labs Staying in remote locations & cannot use the internet every time for studying Group 7 Similar category labels Home disturbance because of high number of family members (100%) Family issues (50%) Home disturbances 2 Cards No functional space at home to study Personal disturbances – e.g., big family, caring for children, church & work commitments 7 Cards Lack of f2f online assessments e.g., online group work Limited opportunity to interact f2f with peers to succeed in the unit Easy to get solutions to quizzes and tests on the internet Lack of student engagement in learning activities No group work incorporated in online learning and assessments No study buddy or partner to study with Struggle to understand math activities and tricky tutorial questions provided online 7 Cards Lack of f2f online assessments e.g., online group work Limited opportunity to interact f2f with peers to succeed in the unit Easy to get solutions to quizzes and tests on the internet Lack of student engagement in learning activities No group work incorporated in online learning and assessments No study buddy or partner to study with p Group 5 Similar category labels Teaching staff Issues (83%) Tutor problem (67% ) Tutor Short fall (67%) Problem with course delivery 6 Cards Typos in lecture notes and examples and solutions having errors cause confusion Lack of mathematics examples from the Pacific context Lack of personal assistance from teaching staff No tutors available weekly for regional students Poor communication and feedback from staff Tutors are less active in the online activities Group 6 Similar category labels Internet challenges (100%) Resources accessibility (80%) Internet issues (67%) Internet challenge 4 Cards A poor internet connection and poor- quality internet Difficulty in downloading large videos file size No access to the computer labs Staying in remote locations & cannot use the internet every time for studying Group 7 Similar category labels Home disturbance because of high number of family members (100%) Family issues (50%) Home disturbances 2 Cards No functional space at home to study Personal disturbances – e.g., big family, caring for children, church & work commitments Group 5 Similar category labels Teaching staff Issues (83%) Tutor problem (67% ) Tutor Short fall (67%) Problem with course delivery 6 Cards Typos in lecture notes and examples and solutions having errors cause confusion Lack of mathematics examples from the Pacific context Lack of personal assistance from teaching staff No tutors available weekly for regional students Poor communication and feedback from staff Tutors are less active in the online activities Group 6 Similar category labels Internet challenges (100%) Resources accessibility (80%) Internet issues (67%) Internet challenge 4 Cards A poor internet connection and poor- quality internet Difficulty in downloading large videos file size No access to the computer labs Staying in remote locations & cannot use the internet every time for studying Group 7 Similar category labels Home disturbance because of high number of family members (100%) Family issues (50%) Home disturbances 2 Cards No functional space at home to study Personal disturbances – e.g., big family, caring for children, church & work commitments Table 4: The findings from the participant’s response to the questionnaire. 6.1 Financial hardship Financial hardship is one of the major challenges faced by the majority of participants during the COVID-19 lockdown. The findings indicate the effects of job displacement on students’ financial situation during an emergency. As reported, domestic workers have suffered from job loss and/or a drop in working hours as one of the negative impacts of COVID-19 (International Labour Organisation, 2020). The issues of parental unemployment and job displacement during COVID-19 have put many families around the world in financial crisis, making it very difficult for them to take care of everyday needs including education. It is evident in this study that participants have identified financial difficulties as the leading cause of their learning challenges which inter alia lead to human stress during COVID-19. 6 Findings And Discussion Page 16/31 Page 16/31 Rate 1 - top challenge 2 3 4 5 6 7 - least challenge Challenge Financial Hardship Motivation Challenge Moodle Issues Lack of f2f interaction Problem with course delivery Internet challenge Home disturbances Rate per cent 59.4 53.1 59.4 50 48.9 56.3 75 Number of participants 19 17 19 16 15 18 24 Since the research sets out to understand first-year mathematics students’ learning challenges during the unexpected shift from f2f to ERT, this section discusses participants’ findings against the literature in order of the seven categories of challenges presented in Tables 3 and 4. The primary purpose of Table 3 is to show how the participants rated the category names that they found affecting their studies the most during the shift. As seen in Table 4, financial hardship is the biggest challenge and the home disturbance is the least challenge during the unexpected shift from f2f to ERT. Table 4 is also reflected the hidden meaning showing in Figure 7. The number of cards indicates how serious the challenges are, meaning that most students encountered a learning challenge during COVID-19. The higher the number of cards in a cluster, the more frequently the challenge is being faced by students during the COVID-19 crisis. 6.3 Moodle issues The effectiveness of online learning depends on the designed and prepared learning material, the lecturer’s engagement in the online environment, and lecturer–student and/or student–student interactions (e.g., Bao 2020; Wu and Liu 2013). In consideration of the list of challenges compiled by participants under this category, it suggests that without having a proper design of the moodle page and a proper training of the moodle page with students-lecturers as end users, the full potential and purpose of establishing such a platform cannot be reached, hence having negative impacts on students’ success. The majority of participants believe that poor audio and lecture/tutorial videos as well as too much information in the course shell can cause confusion. The challenge of writing mathematics online using moodle features is still huge, and the option of alternatives such as uploading snapshots of write-ups is also not feasible keeping in mind the intermitant internet facilities and costly mobile data during emergencies and crises in the South Pacific. Arguably, moodle is expected to be a friendly learning environment for students, teachers and course instructors to develop, disseminate learning materials, and share knowledge through multiple online activities such as forums and chats (Kumar et al., 2020; Sharma 2020). The authors’ experience has confirmed the need for moodle designers to measure usability because it determines the moodle's success based on students’ learning needs. Many teachers have no previous experience in online teaching and although teachers received various types of training during the outbreak, the short-term effects of such training remains arguably minimal. Therefore, the instructor and students should be aware of both the advantages and potential pitfalls of using the latest technological advances like moodle during the shift to ERT. 6.2 Motivational challenge Studying from home commonly requires greater self-discipline and motivation to follow through online lessons, particularly in the earlier period when students are getting used to the new system, which might affect the feeling of an increase in study obligations. On the other hand, lecturers’ unfamiliarities and incompetencies with the new mode of delivery could overload their students with study materials and assignments adding to the demotivate students feeling toward the course (Aristovnik et al. 2020). An emergency switch from f2f to ERT makes the learning experience entirely different and challenging to Page 17/31 maintain intrinsic motivation in students which is even more pronounced in mathematics learning environment The list of challenges that participants did consider under this category has highlighted the importance of putting an effective learning support system in place and the importance of protecting participants’ motivation and willingness to learn during a pandemic crisis. Self-motivation is an indispensable requirement for online learning; however, it seems to be absent from many online students which is a commitment to be fulfilled by the institution (Bozkurt and Sharma 2020). Also many online students feel disconnected to their studies (Choudhury and Pattnaik 2020). After shifting to ERT, many students fall behind and give up as problems in handling a technological medium also seem difficult due to the lack of relevant ICT competencies (Aguliera and Nightingale-Lee 2020; Reddy et al. 2020). Therefore, the attitude change and technological literacy would help them gain confidence in order to succeed in their ERT courses with a lively atmosphere. 6.6 Internet challenge The sudden closing-off of face-to-face educational work, in response to the COVID-19 pandemic, gave teachers and students a strong sense of the difference between f2f and ERT, arguing that online learning can work more effectively in digitally developed countries (Basilaia & Kvavadze, 2020) such as the United States of America and not in most developing countries. In the Pacific Island countries, online learning (as well as blended learning) is sometimes ineffective due to the lack of access to fast, affordable and reliable internet connections (Reddy et al., 2020; Sharma et al., 2020) or even a lack of electricity. This hinders the process of online learning, especially for those who are living in rural as well as marginalised communities (Aristovnik et al. 2020; Wains and Mahmood 2008). Students who access the internet through smartphones are sometimes powerless to take advantage of online learning because a vital amount of online content is not accessible via smartphones. Low-income families mean that access to the internet is occasional provides poor internet connection and poor-quality internet. 6.5 Problem with course delivery Participants were concerned with the lack of support from teaching staff during the ERT, which can link to the delivery mode's remote nature. This includes little time for inter-personal, poor communication, and being less active in the online activities. Such challenges imply a specific pedagogical content knowledge associated with the designing and organising of healthier learning experiences and distinctive learning environments with the help of digital technologies. The pedagogical readiness of university teachers who have little experience in online teaching has become an integral part of any virtual learning. As reported, some of the main difficulties facing university teachers concerning web-based courses arise from the complexity of the instructional situation and shortcomings in planning and organisation (Ching et al. 2018; Ocak 2011). 6.4 Lack of 2f2 interaction There is broad agreement that teachers play a key role in providing high-quality learning opportunities to students and fostering students’ learning (e.g., König et al. 2021). Most HEI in the Pacific region rely heavily on f2f mode for sharing and distributing knowledge, hence, the capacity of the institution to handle the circumstances of unprecedented change to ERT can be a real challenge. For instance, the majority of participants believe that lack of f2f learning interactions and assessment strategies can make things hard for Page 18/31 Page 18/31 them to succeed during the ERT. Similarly, educators found that a lack of student f2f engagement is a primary problem that can cause distraction in effective learning. To overcome this inadequacy, a range of f2f learning support systems should put in place to improve students’ interest in emergency online learning. This includes visual communication such as video group discussion, live and recorded tutorials; strengthen the insitution’s online learning culture and policies; and allowing academics to be heavily involved in facilitating effective online learning activities that have direct positive impacts on students (Beetham and Sharpe 2007). 7 Potential Solutions For Students’ Challenges The novel seven categories of challenges articulated in this research give direction to the types of support that are relevant for addressing issues as a basis for participants’ success in the sudden shift to ERT during the emergency and crisis of COVID-19. Figure 8 presents two heptagons with the top outlining the seven challenges and the bottom presenting the potential solutions. The colour is used to identify the patterns of relationship between the two pentagons. For instance, the ‘financial hardship’ challenge in the top heptagon and the potential solution ‘provide financial support’ in the bottom heptagon are both coloured in light black to show their relationships. While the model governs the challenges and potential solutions for students studying mathematics, it is equally relevant to students from other disciplines. When we look at these major categories from an affirmative perspective, it means that the institution’s level of preparedness during the ERT must be strengthened. For instance, in terms of ‘financial hardship’, the findings indicate that the institution must be ready to compensate students’ internet and technological needs in an emergency. As witnessed in this study, participants considered ‘financial hardship’ and ‘motivational challenge’ as the lead causes of their learning challenges during the erupted shift to ERT due to COVID-19. It emphasises the need to improve the affordability and availability of free access to learning support during the financial and psychological crisis of a pandemic. The findings also reinforce the significant role of moodle literacy, f2f interaction, quality course delivery, quality internet network access, and having a friendly learning environment at home to participants’ success. Figure 8 highlights a new learning support model designed from the insights of the findings of this research. The model suggests that the appropriate way to support first-year Pacific mathematics students learning challenges during any crisis or emergency must be understood from the dynamic interplay between their finances, motivation, online learning literacies, f2f interpersonal interaction, course delivery, internet access, and home environment within a given socio-cultural learning context. This leads to the understanding that participants’ learning challenges are a complex system, meaning that participants’ learning challenges during ERT is made up of different related parts that must be understood within the socio-cultural context in which it is understood and experienced. 6.7 Home disturbances Due to the COVID-19 lockdown, students were required to study from home at the time of social distancing and lockdown. Students had no choice but to accept this unprecedented change in response to fight against the spread of the virus. The challenges under this category reveal that study from home during an emergency or crisis would be much more difficult and challenging for students who live in villages, extended families and crowded houses without any study-friendly environment. This is a common situation with Pacific students and families from the rural and low socio-economic backgrounds. Therefore the availability of Page 19/31 different kinds of home infrastructure is needed to ensure efficient study. The potential solutions for all the seven challenges emerged from this study based on participants’ card sorting findings are demonstrated in Figure 7. 7 Potential Solutions For Students’ Challenges As demonstrated by bottom heptagon of Figure 8, participants’ challenges can be appropriately addressed in a more interconnected and multidimensional system, It means that people within the respective context, whether at home or in HEI, should be the catalyst for change and driver of students’ success during the pandemic crisis and emergencies. 8 Conclusion Overall, this study shows that the unexpected shift from f2f to ERT due to COVID-19 has affected first-year mathematics students to experience a range of challenges including financial hardship, motivational challenge, moodle issue, lack of f2f interaction, course delivery problem, internet challenge, and home disturbances. These findings emphasise that if these students’ challenges are not fully understood in the Page 20/31 Page 20/31 context of their own socio-cultural contexts, it may erode their motivation and confidence which can impact their overall academic performance. The paper's arterial strength lies in its ability to effectively introduce card sorting in the field of educational research as a provider of new learning analytics for quality learning in mathematics and desirable learning outcomes from a range of known and unknown inputs. The use of card sorting, for the first time, to explore the topic adds new i important connection between card sorting, HEI, mathematics, quality le crisis or emergency such as COVID-19 pandemic. Findings from this stu contribution to the understanding of the challenges faced by first-year P complex system within a given socio-cultural learning context. This prov complex way in which first-year mathematics students operate in their o given pandemic. In particular, the combination of students’ social and c with the institution’s learning support system must go hand in hand for significance of using card sorting as a new methodology in the field of e the students’ challenges from their own perspectives and to design a m model for emergencies and crises. From a wider perspective, the unique the field of educational research and can be utilised in a number of area and other contextualised learning resources and support models. The use of card sorting, for the first time, to explore the topic adds new insights into the literature on the important connection between card sorting, HEI, mathematics, quality learning, and student retention during a crisis or emergency such as COVID-19 pandemic. Findings from this study also offer, for the first time, a contribution to the understanding of the challenges faced by first-year Pacific mathematics students as a complex system within a given socio-cultural learning context. This provides a fresh perspective into the complex way in which first-year mathematics students operate in their own Pacific learning contexts in a given pandemic. Data availability statement Data availability statement The datasets produced for this study are available on request to the corresponding author. ts produced for this study are available on request to the corresponding author 8 Conclusion In particular, the combination of students’ social and cultural learning backgrounds together with the institution’s learning support system must go hand in hand for best results. The study highlights the significance of using card sorting as a new methodology in the field of educational research to understand the students’ challenges from their own perspectives and to design a more student-oriented learning support model for emergencies and crises. From a wider perspective, the unique methodology has a good scope in the field of educational research and can be utilised in a number of areas such as design of online courses and other contextualised learning resources and support models. The findings from this study would benefit our understanding of the challenges faced by first-year mathematics students at a HEI in the Pacific. It highlights the need for more in-depth future exploration of the seven aspects of the Pacific learning support system proposed in the study. Such a study can explore and evaluate each challenge's main causes and discovery measures from students’ own perceptions and experiences. The use of Pacific research approaches, such as ‘talanoa’ (talking) (Paea et al. 2020) can produce a lot of rich and deep knowledge about the research topic using qualitative data. An extension of the research setting to the wider Pacific region via the USP’s regional campuses would enhance understanding the topic from the dynamic nature of the South Pacific cultural diversity. Author Contributions The authors of this paper who agreed to be accountable for all aspects of this study in confirming that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved honesty. We provided substantial contributions to the design of this study, clarifying and interpretation of the data, and revised it critically for intellectual content. Conflict of interest The authors declare that they have no conflict of interest Funding Funding information is not applicable / No funding was received. Page 21/31 Acknowledgments A special thank goes out to all participants who have offered their contribution to this study. Thank you for sharing your time and experience. 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Figures Figure 1 Participant pathway through f2f OCS with physical cards in real-time. Figure 1 Participant pathway through f2f OCS with physical cards in real-time. Participant pathway through f2f OCS with physical cards in real-time. Page 26/31 Page 26/31 Page 26/31 Figure 3 Shows the portion along the right edge of Figure 2 and indicates how many participants agree with each paired combination of cards. Shows the portion along the right edge of Figure 2 and indicates how many participants agree with each paired combination of cards. Figure 4 Determining the optimal number of categories. a) The scree plot for the initial variables. B) The scree plot analysis. Figure 2 The similarity matrix displays how many participants agree with each pair combination of cards. The algorithm attempts to cluster similar cards along the right edge of the matrix. Page 27/31 Page 27/31 Page 27/31 Page 27/31 Figure 3 Shows the portion along the right edge of Figure 2 and indicates how many participants agree with each paired combination of cards. Figure 3 Figure 3 Figure 4 Determining the optimal number of categories. a) The scree plot for the initial variables. B) The scree plot analysis. Determining the optimal number of categories. a) The scree plot for the initial variables. B) The scree plot analysis. Determining the optimal number of categories. a) The scree plot for the initial variables. B) The scree plot analysis. Page 28/31 Figure 5 Figure 5 The graph of stress against the number of dimensions (a) and R2 versus the number of dimensions (b) The graph of stress against the number of dimensions (a) and R2 versus the number of dimensions (b) The graph of stress against the number of dimensions (a) and R2 versus the number of dimensions (b) Page 29/31 Page 29/31 Figure 6 The 3DCV of the clustering results in Table 1. Using 3DCV produced seven-cluster solutions with each cluster shown in different colours. These polygon groups can be interpreted as possible categories for IA. The 3DCV of the clustering results in Table 1. Using 3DCV produced seven-cluster solutions with each cluster shown in different colours. These polygon groups can be interpreted as possible categories for IA. For clear visualisation, Bar graph of Figure 2 with the number of cards in each polygon. Figure 7 Figure 7 For clear visualisation, Bar graph of Figure 2 with the number of cards in each polygon. For clear visualisation, Bar graph of Figure 2 with the number of cards in each polygon. Page 30/31 Page 30/31 Figure 8 Figure 8 Potential solutions for participants’ challenges Potential solutions for participants’ challenges Page 31/31 Page 31/31
https://openalex.org/W4366817211	https://journals.plos.org/ploscompbiol/article/file?id=10.1371/journal.pcbi.1011055&type=printable	English	null	Shape-driven deep neural networks for fast acquisition of aortic 3D pressure and velocity flow fields	PLOS computational biology/PLoS computational biology	2,023	cc-by	10,627	PLOS COMPUTATIONAL BIOLOGY PLOS COMPUTATIONAL BIOLOGY Editor: Daniel A. Beard, University of Michigan, UNITED STATES Received: November 4, 2022 Accepted: March 28, 2023 Published: April 24, 2023 Received: November 4, 2022 Accepted: March 28, 2023 Published: April 24, 2023 Copyright: © 2023 Pajaziti et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Copyright: © 2023 Pajaziti 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. RESEARCH ARTICLE Shape-driven deep neural networks for fast acquisition of aortic 3D pressure and velocity flow fields Endrit PajazitiID1, Javier Montalt-Tordera1, Claudio Capelli1, Raphae¨l Sivera1, Emilie Sauvage1, Michael Quail2, Silvia Schievano1, Vivek Muthurangu1 Endrit PajazitiID1, Javier Montalt-Tordera1, Claudio Capelli1, Raphae¨l Sivera1, Emilie Sauvage1, Michael Quail2, Silvia Schievano1, Vivek Muthurangu1 1 University College London, Institution of Cardiovascular Science, London, United Kingdom, 2 Great Ormond Street Hospital, Cardiac Unit, London, United Kingdom a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 * endrit.pajaziti.13@ucl.ac.uk * endrit.pajaziti.13@ucl.ac.uk * endrit.pajaziti.13@ucl.ac.uk a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 Abstract Computational fluid dynamics (CFD) can be used to simulate vascular haemodynamics and analyse potential treatment options. CFD has shown to be beneficial in improving patient outcomes. However, the implementation of CFD for routine clinical use is yet to be realised. Barriers for CFD include high computational resources, specialist experience needed for designing simulation set-ups, and long processing times. The aim of this study was to explore the use of machine learning (ML) to replicate conventional aortic CFD with auto- matic and fast regression models. Data used to train/test the model consisted of 3,000 CFD simulations performed on synthetically generated 3D aortic shapes. These subjects were generated from a statistical shape model (SSM) built on real patient-specific aortas (N = 67). Inference performed on 200 test shapes resulted in average errors of 6.01% ±3.12 SD and 3.99% ±0.93 SD for pressure and velocity, respectively. Our ML-based models performed CFD in 0.075 seconds (4,000x faster than the solver). This proof-of-concept study shows that results from conventional vascular CFD can be reproduced using ML at a much faster rate, in an automatic process, and with reasonable accuracy. OPEN ACCESS Citation: Pajaziti E, Montalt-Tordera J, Capelli C, Sivera R, Sauvage E, Quail M, et al. (2023) Shape- driven deep neural networks for fast acquisition of aortic 3D pressure and velocity flow fields. PLoS Comput Biol 19(4): e1011055. https://doi.org/ 10.1371/journal.pcbi.1011055 Editor: Daniel A. Beard, University of Michigan, UNITED STATES Introduction Competing interests: The authors have declared that no competing interests exist. Computational fluid dynamics (CFD) has significant potential in cardiovascular settings, with applications including modelling of complex flow patterns and non-invasive estimation of vas- cular pressure [1]. Importantly, several studies have validated CFD measurements against con- ventional clinical methods such as 4D cardiovascular magnetic resonance (CMR) imaging and catheter-based pressure measurements [2, 3]. A major strength of CFD lies in situations where it is difficult to acquire data with conventional clinical means. For example, studies have shown CFD can be used to assess the haemodynamic response to exercise as a non-invasive alternative to cardiac catheterisation with exercise or pharmacological stress [4]. Another example is the use of CFD for predicting the haemodynamic response to specific interventions, such as aortic stenting in patients with coarctation [5]. Furthermore, there is increasing inter- est in exploring CFD-derived metrics for risk stratification of patients. For example, Qiu et al. demonstrated that CFD derived helical flow patterns were associated with significantly increased risk of abdominal aortic aneurysm rupture [6]. Thus, integration of CFD into clini- cal settings may have important ramifications when used: (i) for detailed assessment of patient-specific haemodynamics in response to stress or interventions, and (ii) to estimate CFD-derived indices for supporting clinical decision-making and risk-stratification. Despite potential benefits, CFD is still not integrated into routine clinical practice. This is mainly due to long computation times, the requirement for large amounts of processing power, and the need for an experienced engineer to set up simulations correctly [7]. Recently, machine learning (ML) models have been successful in replacing time-consuming or compu- tationally intensive tasks, such as medical image segmentation [8]. Similarly, the use of ML for speeding up cardiac CFD tasks is also becoming increasingly studied [9, 10]. The main chal- lenges of training ML models using CFD data include: (i) poor availability of clinical data, (ii) unstructured meshes without point correspondence, and (iii) large meshes and resultant CFD flow fields. To overcome these problems, we used statistical shape modelling and dimensional- ity reduction techniques to produce dimensionality-reduced representations of both aortic shape and flow fields, and to enable creation of large amounts of synthetic training data [11, 12]. This approach relies on the fact that in the past it has been show haemodynamic flow structures can be regressed from shape features [13]. PLOS COMPUTATIONAL BIOLOGY Deep neural networks for fast aortic 3D pressure and velocity flow fields This is a PLOS Computational Biology Methods paper. Medical Research, received by ES (GN2572), European Research Council, received by SS (ERC- 2017-StG-757923). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Medical Research, received by ES (GN2572), European Research Council, received by SS (ERC- 2017-StG-757923). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Author summary In the clinical management of pediatric disease (namely congenital heart defects), the indications for ‘when’ and ‘how’ to intervene are often unclear. It has been found that hae- modynamic modelling tools such as computational fluid dynamics (CFD) simulations are useful in assisting clinicians and surgeons to better understand patient conditions and establish any potential risk factors. While this tool remains useful in a research capacity, its separation from clinical settings is an ongoing hindrance which prevents the full adop- tion of CFD in healthcare. The translation of CFD towards clinics is a continuous chal- lenge, due to large time, computational and human resource requirements for running simulations. The application of machine learning (ML) for exploring potential methods to transform conventional CFD into clinically-suitable models is a recent phenomenon which is gaining significant momentum. Data Availability Statement: All code related to the study can be found in the GitHub repository CFD_Machine_Learning: https://github.com/ EndritPJ/CFD_Machine_Learning. Funding: We would like to acknowledge the following funders: UK Research and Innovation, received by SS (MR/S032290/1), Heart Research UK, received by VM (RG2661/17/20), British Heart Foundation, received by SS (NH/18/1/33511), the Engineering and Physical Sciences Research Council, received by SS (EP/N02124X/1), Action 1 / 20 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1011055 April 24, 2023 Introduction Our primary aim was to show the feasibility of a shape-driven ML model for the accurate estimation of 3D CFD flow fields in a population of aortas with challenging shape features. The main aims of this study were: (i) to create a large synthetic cohort of 3D aortas based on real clinical cases with complex anatomies, (ii) to train ML models to predict aortic pressure and velocity fields by representing unstructured/large data types with low-dimensional vectors, and (iii) to compare results between our fast/automatic ML-based CFD solution and our con- ventional CFD method on both synthetic and real aortic cases. mesh-preprocessing pipeline. The aorta segmentation of each subject is re-meshed and smoothed in an automatic pipeline. g of the inlets/outlets and head & neck vessels. Fig 1. Segmentation and mesh-preprocessing pipeline. The aorta segmentation of each subject is re-meshed and smoothed in an automatic pipeline. This is followed by clipping of the inlets/outlets and head & neck vessels. https://doi.org/10.1371/journal.pcbi.1011055.g001 retrospectively collected data was approved by the local research ethics committee, and written consent was obtained from all subjects/guardians (Ref: 06/Q0508/124). Images for each subject were segmented and converted into surface meshes (Fig 1). This was followed by remeshing and smoothing using functions from the vascular modelling toolkit (VMTK) [14]. All geome- tries were aligned in the same local space and orientation through rigid registration using an iterative closest point algorithm in VMTK [15]. This ensured that shape modelling was not affected by any spatial misalignment. Surfaces were manually clipped above the aortic root for the inlet, and at the diaphragm for the outlet. An SSM was then built using these clipped aortic surfaces, using an approach previously described by Bruse et al [16]. g pp p y y The package Deformetrica 4 was used to build the SSM [17]. First, an average aortic shape (surface template) was computed, containing 2,541 nodes (Fig 2). A volume template was also created by meshing the surface template with tetrahedral elements (29,000 nodes). Each sub- ject could then be described as a non-linear deformation of 3D ambient space, relative to the template (Fig 2). In this case, each deformation is fully parameterised by a paired set of 3D control points (qi)i=1,. . .n and 3D momenta vectors (μi)i=1,. . .n using a Gaussian kernel of width σ which we set as 10 mm. The number and location of control points were optimised (n = 172) by initialising the model with a high resolution control point grid (n = 500) and truncating points which were observed to have little influence on the deformation (low variance). The final computed 3D deformations for a given subject were represented by a deformation vector with 516 coefficients—the number of control points (172) multiplied by the number of defor- mation directions (3). The deformation vectors for the whole population were collected in the 2D matrix M [67, 516], which was decomposed using principal component analysis (PCA) in order to identify low-dimensional deformations which account for most of the variance. Statistical shape modelling The dataset used for development of the statistical shape model (SSM) consisted of cardiac and respiratory gated steady state free precession CMR images (N = 67) from patients previously diagnosed with coarctation of the aorta (CoA). All patients were post-surgical repair, asymp- tomatic and underwent CMR imaging at a mean age of 22.4 ±6.2 years. The use of 2 / 20 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1011055 April 24, 2023 PLOS COMPUTATIONAL BIOLOGY Deep neural networks for fast aortic 3D pressure and velocity flow fields Fig 1. Segmentation and mesh-preprocessing pipeline. The aorta segmentation of each subject is re-meshed and smoothed in an automatic pipeline. This is followed by clipping of the inlets/outlets and head & neck vessels. https://doi.org/10.1371/journal.pcbi.1011055.g001 This required standardisation for each column in M (i.e. removal of the mean and scaling to unit variance). Following this, singular value decomposition (SVD) was performed, with M being decomposed into three matrices: M = USVT. The V transpose matrix [516, 516] contained the principal component axes or PCA modes, S is a rectangular diagonal matrix [67, 516] where the singular values on the diagonal can be used to calculate the variance explained by the PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1011055 April 24, 2023 3 / 20 PLOS COMPUTATIONAL BIOLOGY Deep neural networks for fast aortic 3D pressure and velocity flow fields Fig 2. Mesh registration with SSM. An example aortic shape approximation using our SSM is shown. Individual surface or volumes can be reconstructed using a mean aortic shape and applied deformation field initialised on a set of control points (n = 172). https://doi.org/10.1371/journal.pcbi.1011055.g002 Fig 2. Mesh registration with SSM. An example aortic shape approximation using our SSM is shown. Individual surface or volumes can be reconstructed using a mean aortic shape and applied deformation field initialised on a set of control points (n = 172). https://doi.org/10.1371/journal.pcbi.1011055.g002 https://doi.org/10.1371/journal.pcbi.1011055.g002 https://doi.org/10.1371/journal.pcbi.1011055.g002 associated PCA modes, and the matrix product US [67, 516] contains the projection (weights) of each subject onto each PCA mode. It was found that the first 35 PCA modes were capable of approximating 99% of the variance in M. This meant that specific aortic shapes could be repre- sented by a lower-dimensional deformation vector with 35 coefficients rather than 516 (almost 15 times reduction in the size). Using the SSM, new synthetic aortic shapes could then be created using synthetic lower dimensional deformation vectors. Specifically, each of the 35 coefficients in a synthetic vector was generated by randomly sampling a Gaussian distribution (within 2 standard deviations) based on the distribution of weights in the US matrix. Following concatenation of all the lower dimensional deformation vectors, the matrix X [3000, 35] was transformed into a matrix L [3000, 516] by matrix multiplication (L = XSVT), thus reversing PCA and mapping the matrix X onto the original axes. Standardisation was then reversed in all columns of L using the previ- ously computed standard deviations and means in the M matrix. The deformation matrix L was then reshaped into a 3D momenta matrix [3000, 172, 3] and applied onto the aortic sur- face and volume template using Deformetrica, thus generating a surface and volume mesh for each new synthetic subject. Since all new meshes are derived from the same template, all syn- thetic aortas contained the same number of nodes/elements. Additionally, nodes can be thought to be lying within spatially correspondent locations within each aorta (see S1 Fig). This was vital for enabling the dimensionality reduction of derived flow fields, as described in later sections. The new synthetic population (n = 3000) was compared to the original population (n = 67) by computing geometric properties of the shapes based on a centreline approach. Mean cen- treline lengths and diameters were computed. Mean torsion is used to express how sharply the centreline is twisting in space. The parameter tortuosity describes the length ratio between the centreline and a rectilinear line between the endpoints. All parameters were computed using implementations within VMTK, as described by Piccinelli et al. [18]. PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1011055 April 24, 2023 4 / 20 PLOS COMPUTATIONAL BIOLOGY Deep neural networks for fast aortic 3D pressure and velocity flow fields Computational fluid dynamics pipeline Volume meshes previously generated with the SSM were unsuitable for CFD computation. This was primarily because low mesh skewness could not be guaranteed, and remeshing was not an option since nodes were to be preserved in order to maintain point correspondence (see S1 Fig). Therefore, separate meshes solely for CFD computation were built, starting from the surface of each aorta. Firstly, each surface was extended by 40mm at the inlet (Fig 3). This was done to produce a flat and circular inlet upon which a velocity profile could be uniformly applied. Extending the inlet further than 40mm was avoided in order to reduce the likelihood of surface self-intersection. Following this, volume meshing with tetrahedral elements was per- formed [19] (400,000 cells on average). Element/node counts were deemed to be in satisfac- tory ranges after a mesh sensitivity analysis (see S2 Fig). CFD (Fluent, Ansys Technologies) was performed on all 3,000 synthetic cases. The same boundary conditions were applied to each simulation, as part of adopting a simple model which would reliably converge for all subjects. Laminar, steady-state flow conditions were enforced. An inlet velocity of 1.3 m/s, corresponding to an average ascending aortic flow rate at peak systole, was set [20, 21]. A velocity boundary condition was preferred to a volumetric flow rate, since it is invariant to any differences in inlet surface area between subjects. Outlet gauge pressure was fixed at 0 Pa. Standard non-slip conditions were applied at the wall, and the fluid was assumed to be Newtonian with density and dynamic viscosity equal to 1,060 kg/ m3 and 0.004 Pas, respectively [22]. The set-up and simulation of all 3,000 cases was fully automated. https://doi.org/10.1371/journal.pcbi.1011055.g003 Machine learning Data interpolation and principal component analysis. As CFD was performed on large unstructured meshes with inconsistent numbers of nodes/elements between cases, point corre- spondence had to be restored prior to PCA-based dimensionality reduction of the flow fields (needed for easier model training). This was done using the volume meshes previously gener- ated with the SSM by ‘shooting’ on the template. Since each of these ‘SSM volume meshes’ Fig 3. CFD Pipeline. Surfaces have flow extensions added before volume meshing. The same simulation set-up is applied to the final volume mesh for each synthetic subject. Fig 3. CFD Pipeline. Surfaces have flow extensions added before volume meshing. The same simulation set-up is applied to the final volume mesh for each synthetic subject Fig 3. CFD Pipeline. Surfaces have flow extensions added before volume meshing. The same simulation set-up is applied to the final volume mesh for each synthetic subject. https://doi.org/10.1371/journal.pcbi.1011055.g003 https://doi.org/10.1371/journal.pcbi.1011055.g003 5 / 20 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1011055 April 24, 2023 PLOS COMPUTATIONAL BIOLOGY Deep neural networks for fast aortic 3D pressure and velocity flow fields inherited its properties from the template, relative nodal positions were preserved. Conse- quently, pressure/velocity data for all subjects were re-sampled from unstructured CFD meshes onto SSM volume meshes using a Voronoi kernel (padding of 5 mm, grid resolution of 1,000,000 voxels) in the software Paraview (Fig 4). This resulted in 3,000 newly resampled pressure/velocity fields, with all subjects containing 29,000 nodes in point correspondence. The data was concatenated into a feature vector and PCA was applied to reduce dimensional- ity. We aimed to capture 99% of variance with as few PCA modes as possible. Deep neural network architecture. The architecture we adopted was a standard sequen- tial, fully-connected deep neural network (DNN) with independent networks for pressure and velocity. The input for the model was the lower dimensional deformation vector, which is also referred to as a ‘shape vector’. The outputs of the trainable part of the model were the pressure/ velocity PCA scores (reduced order CFD field), referred to as a ‘pressure/velocity vector’. A non-trainable inverse PCA layer (implemented in Keras using a lambda layer) serves to recon- struct the pressure/velocity vector into the full 3D flow field with 29,000 nodes (see Fig 5). Rec- tified linear units (ReLU) were used in each hidden layer. Linear activation functions were set at the output. Model implementation was done using Keras and TensorFlow 2.0. Machine learning Deep neural network training. Models were built separately for predicting the static pres- sure and the velocity-magnitude. The loss function used for training was the mean absolute error (MAE), computed on the entire 3D flow field (i.e. after inverse PCA) rather than on the output pressure/velocity vector. This provides a more granular measure of error and effectively weights the importance of each PCA mode in the network according to the amount of variance it explains. Model optimisation was carried out using the Adam optimiser [23]. The training Fig 4. CFD data interpolation. CFD results are interpolated onto a point-correspondence mesh (generated by the SSM), thus restoring node concordance. Fig 4. CFD data interpolation. CFD results are interpolated onto a point-correspondence mesh (generated by the SSM), thus restoring node concordance ata interpolation. CFD results are interpolated onto a point-correspondence mesh (generated by the SSM), thus restoring nod Fig 4. CFD data interpolation. CFD results are interpolated onto a point-correspondence mesh (generated by the SSM), thus restoring node concordance. https://doi.org/10.1371/journal.pcbi.1011055.g004 https://doi.org/10.1371/journal.pcbi.1011055.g004 6 / 20 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1011055 April 24, 2023 PLOS COMPUTATIONAL BIOLOGY Deep neural networks for fast aortic 3D pressure and velocity flow fields Fig 5. DNN general architecture. The general sequential, fully-connected DNN set-up used to build both pressure and velocity predictors (’CFD vector’ can be either pressure or velocity PCA vectors). Fig 5. DNN general architecture. The general sequential, fully-connected DNN set-up used to build both pressure and velocity predictors (’CFD vector’ can be either pressure or velocity PCA vectors). https://doi.org/10.1371/journal.pcbi.1011055.g005 dataset was composed of 2800 randomly selected subjects, with the remaining (200) going into the test set. Hyperparameter tuning was conducted using 5-fold cross-validation to find model settings which generalise well for unseen data. For this, the training dataset was split into 5 groups (580 subjects each). The model was trained and tested five times, with each group taking turns to act as the validation set while the remaining four were used for training. The average validation error for all five training runs was used to represent the performance of that model. This pro- cess was repeated for 1,000 model configurations, which were sampled using a tree-structured Parzen estimator (TPE) algorithm [24]. The number of hidden layers, number of hidden layer neurons and initial learning rate were all explored. Batch size and epochs were set at 32 and 50, respectively. NAEði; jÞ ¼ jTruei; j Predi; jj RangeðTruejÞ 100% ð1Þ Machine learning Hyperband pruning was used to terminate early training rounds if the model was deemed to be poorly fitting the validation data. After completing hyperparameter tuning, the best model was retrained for 1000 epochs using a larger portion of the training set (2,600 sub- jects) with 200 remaining for validation. Model training lasted 1,000 epochs and training/vali- dation loss was monitored to prevent overfitting. Model training was performed using an Nvidia GTX 1080Ti graphics card. Model evaluation. Once trained, the model was evaluated on the test set of synthetic aor- tas (n = 200). Absolute errors were computed for every node in all test cases by comparing the prediction value (ML) to the ground-truth value (CFD). Errors were then normalised accord- ing to subject CFD data range, as detailed in Liang et al. [11]. Normalisation was necessary to enable direct comparison between individual cases and also between CFD metrics, since pres- sure/velocity ranges widely differed per subject. Eq 1 details how normalised absolute error (NAE) is computed for either pressure or velocity at a node i, belonging to a subject j. Truei,j is the CFD nodal pressure/velocity value. Predi,j is the ML nodal pressure/velocity value. Range (Truej) is the difference between the maximum and minimum values in the CFD flow field for subject j. NAEði; jÞ ¼ jTruei; j Predi; jj RangeðTruejÞ 100% ð1Þ ð1Þ 7 / 20 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1011055 April 24, 2023 PLOS COMPUTATIONAL BIOLOGY Deep neural networks for fast aortic 3D pressure and velocity flow fields Mean node errors (MNAEN) were computed by averaging NAE values across the popula- tion for each node (n = 29,000). These were then plotted on the template mesh points in order to better visualise the magnitude of these errors with respect to their location. However, since NAE values are absolute errors, this provides no insight regarding any systematic over or under-estimation during model inference. Therefore, a Bland-Altman plot was used to exam- ine the bias and limits of agreement of the pressure and velocity DNNs. This was done for the overall aorta and for three separate regions; ascending aorta, transverse arch and descending aorta (anatomically defined). Mean subject errors (MNAES) were computed by averaging NAE values in each subject (n = 200). Cases with the best, median and worse mean subject error values were compared. Machine learning A single population error for both pressure and velocity was given by averaging all MNAES val- ues. The relationship between shape mode scores and subject error (MNAES) was investigated with scatter plots and assessed using Pearson R coefficients and p values (p = 0.05 considered significant). In addition to evaluation of the models on the test set (n = 200), the models were also tested on real, patient-specific aortas with previously repaired CoA (n = 10), completely unseen from the SSM and the DNN. This was done in order to validate the robustness of the models for inferring accurate flow fields on real subjects outside our synthetic training/testing sets. CMR images of each patient were segmented. Surfaces were approximated by non-rigid registration (applying deformations on the template) using the SSM. Deformation matrices for each case were decomposed into PCA shape vectors and passed as inputs into the DNN models. Pressure and velocity-magnitude fields were inferred for each subject. Following this, all ten predictions were compared to CFD flow fields computed using both SSM derived geometries and real geometries. Comparison between ML and SSM derived flow fields were made using MNAES as previ- ously described. However, it was not possible to compare the predicted and real CFD flow fields with node-based metrics due to the lack of node-to-node correspondence and exact sur- face matching. Therefore, we used a gradient-based approach to enable direct comparison of pressure/velocity flow fields without shape correspondence. Subject centrelines were used to calculate plane-averaged pressure/velocity gradients at 99 locations over the length of the aorta (Fig 6). To compare gradients, the Fre´chet distance (FD) was used. The FD is a measure of similarity between two point-sets of curves, taking into account the location and ordering of the curve coordinates. Intuitively, it can be thought of as the shortest possible distance between two observers traversing different paths while remaining connected. An advantage of using the FD is that it does not neglect sharp spikes or differences between gradients, which some other metrics may diminish through averaging. Additionally, since FD is not a percentage error, it does not emphasise errors where values are close to zero (such as at the very end of the descending aorta in pressure flow fields). An algorithmic implementation for computing FD as described by Eiter et al. was used to calculate this metric [25]. Statistical shape modelling PCA decomposition was performed on the deformation matrices (momenta) computed by sta- tistical shape modelling. The first, second and third PCA modes captured 29.8%, 13.2% and 10.1% (total 53.1%) of the variability, respectively. Mode 1 relates to overall vessel size, mode 2 relates to ascending arch angulation/diameter, and mode 3 describes rounded versus triangu- lar arches. After PCA decomposition, 99% of the variance in the momenta could be repre- sented with the first 35 modes. Some examples of the 3,000 synthetic subjects produced by PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1011055 April 24, 2023 8 / 20 PLOS COMPUTATIONAL BIOLOGY Deep neural networks for fast aortic 3D pressure and velocity flow fields Fig 6. CFD gradient extraction method. Using subject centrelines, 99 plane-averaged pressure or velocity points along the length of the aorta are extracted by sampling the 3D flow fields. The origin is always the aortic root (excluding the extension). https://doi.org/10.1371/journal.pcbi.1011055.g006 Fig 6. CFD gradient extraction method. Using subject centrelines, 99 plane-averaged pressure or velocity points along the extracted by sampling the 3D flow fields. The origin is always the aortic root (excluding the extension). https://doi.org/10.1371/journal.pcbi.1011055.g006 Fig 6. CFD gradient extraction method. Using subject centrelines, 99 plane-averaged pressure or velocity points along the length of the aorta are extracted by sampling the 3D flow fields. The origin is always the aortic root (excluding the extension). https://doi.org/10.1371/journal.pcbi.1011055.g006 randomly sampling and combining 35 PCA mode scores are shown in Fig 7. Anatomical char- acteristics of the synthetic aortas (length, diameter, tortuosity and torsion) were found to be statistically similar to those of the real patient cohort (see Table 1). Model architecture The model input layer size was set at 35 (number of shape modes). Output layer sizes were set at 20 and 55 for pressure and velocity, respectively (number of pressure/velocity modes). Hyperparameter tuning using cross-validation was performed 1,000 times to search for the optimal learning rate, number of neurons and number of layers, with tuning taking 4 hours per model. Pressure and velocity model architectures as a result of the optimisation process are shown in S4 Fig. Training data After CFD was computed on all cases (n = 3000), values were interpolated from high resolu- tion meshes onto lower-resolution grids in point correspondence. The mean loss in accuracy due to interpolation was found to be 0.056% ±0.027 and 0.849% ±0.247 for pressure and veloc- ity-magnitude, respectively. This was computed by calculating the mean percentage error in centreline pressure and velocity gradients for all cases (n = 3000) and averaging the results. The data post-interpolation was used as the ‘ground-truth’ training and testing sets. PCA decomposition of the pressure and velocity training data matrices ([2800, 29000] each) was then performed, following standardisation. After PCA decomposition, 99% of the standardised pressure variance could be captured with 20 modes. Only 87% of the standard- ised velocity variance could be captured with 55 modes, and it was felt that adding more modes to capture greater variance was not feasible due to massively diminishing returns. Sub- ject errors resulting from PCA decomposition were tested on the 200 test cases (unseen by the PCA model). Average MNAES in pressure and velocity fields were found to be 1.46% ± 0.59 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1011055 April 24, 2023 9 / 20 PLOS COMPUTATIONAL BIOLOGY Deep neural networks for fast aortic 3D pressure and velocity flow fields Fig 7. Modes of deformation. Left: first three modes of deformation from the SSM (SD = standard deviation). Right: examples of synthetic post-CoA aortas from the test set (using combinations of all 35 shape modes). https://doi org/10 1371/journal pcbi 1011055 g007 Fig 7. Modes of deformation. Left: first three modes of deformation from the SSM (SD = standard deviation). Right: examples of synthetic post-CoA aortas from the test set (using combinations of all 35 shape modes). https://doi.org/10.1371/journal.pcbi.1011055.g007 https://doi.org/10.1371/journal.pcbi.1011055.g007 https://doi.org/10.1371/journal.pcbi.1011055.g007 SD and 2.70% ±0.49 SD. The reconstructed test cases with the highest MNAES for pressure and velocity (4.32% and 4.93%, respectively) are shown in S3 Fig. Deep neural networks for fast aortic 3D pressure and velocity flow fields Deep neural networks for fast aortic 3D pressure and velocity flow fields Fig 8. Nodal error analysis. Left: Distribution of mean nodal errors (MNAEN), computed on the test set (n = 200). Errors are absolute values and are projected on the template aorta. Right: Bland-Altman plots for the overall aorta. Normalised error (%) refers to the NAE of each node in every test case, without taking the absolute value (n = 5,800,000). Only 1,000 randomly selected points were drawn to improve graph readability. https://doi.org/10.1371/journal.pcbi.1011055.g008 Fig 8. Nodal error analysis. Left: Distribution of mean nodal errors (MNAEN), computed on the test set (n = 200). Errors are absolute values and are projected on the template aorta. Right: Bland-Altman plots for the overall aorta. Normalised error (%) refers to the NAE of each node in every test case, without taking the absolute value (n = 5,800,000). Only 1,000 randomly selected points were drawn to improve graph readability. Fig 8. Nodal error analysis. Left: Distribution of mean nodal errors (MNAEN), computed on the test set (n = 200). Errors are absolute values and are projected on the template aorta. Right: Bland-Altman plots for the overall aorta. Normalised error (%) refers to the NAE of each node in every test case, without taking the absolute value (n = 5,800,000). Only 1,000 randomly selected points were drawn to improve graph readability. https://doi.org/10.1371/journal.pcbi.1011055.g008 https://doi.org/10.1371/journal.pcbi.1011055.g008 conventional CFD took 5 minutes on average for convergence, demonstrating an approxi- mate 4,000x speed-up with ML. Node errors. Average node prediction errors (MNAEN) were computed for all nodes (n = 29,000). Fig 8 (left) shows these values projected onto the template (average position of the nodes), allowing for the locations of the highest absolute errors to be assessed. Pressure errors were observed to be lower in the descending aorta, with the highest errors situated in the transverse arch. Velocity errors were notably more prevalent in the underside of the arch and descending aorta. The maximum MNAEN was observed to be 12.46% and 14.86% for pressure and velocity, respectively. Bland-Altman analysis showed negligible prediction biases for the overall aorta (Fig 8, right) and within selected regions (S5 Fig). Bland-Altman biases were found to be 0.19% and 0.28% for pressure and velocity, respectively. The limits of agreement were found to be mar- ginally wider for pressure when compared to velocity (15.65% vs 12.89%, respectively). Subject errors. Deep neural networks for fast aortic 3D pressure and velocity flow fields The population error for pressure and velocity was 6.01 ±3.12% SD and 3.99 ±0.93% SD, respectively. The test cases with the best, median and worst subject error (MNAES) are shown in Fig 9 with corresponding pressure/velocity gradients. The maximum MNAES for pressure and velocity were found to be 23.60% and 8.07%, respectively. The mini- mum MNAES for pressure and velocity were found to be 1.54% and 1.91%, respectively. The relationship between pressure/velocity MNAES and shape mode coefficients is pre- sented in Fig 10. The second and third mode showed statistically significant correlations with velocity prediction MNAES, with the third shape mode showing the highest correlation (R= −0.31). No significant correlations were found between any other shape mode and MNAES. Model predictive performance Pressure and velocity-magnitude fields were computed on the test set (n = 200) using the trained DNNs. Inference took 0.075 seconds per subject for both DNNs. In comparison, Table 1. Comparison of aorta dimensions between original real cohort (n = 67) and synthetic cohort (n = 3000). Mean diameter (mm) Length (mm) Tortuosity Mean torsion Real cohort average (n = 67) 19.74 ±1.29 257.3 ±29.88 2.21 ±0.39 0.0044 ±0.061 Synthetic cohort average (n = 3000) 20.12 ±1.32 258.5 ±23.08 2.19 ±0.35 0.0012 ±0.053 p value (Welch’s t-test) 0.25 0.75 0.74 0.65 Table 1. Comparison of aorta dimensions between original real cohort (n = 67) and synthetic cohort (n = 3000). Mean diameter (mm) Length (mm) Tortuosity Mean torsion Real cohort average (n = 67) 19.74 ±1.29 257.3 ±29.88 2.21 ±0.39 0.0044 ±0.061 Synthetic cohort average (n = 3000) 20.12 ±1.32 258.5 ±23.08 2.19 ±0.35 0.0012 ±0.053 p value (Welch’s t-test) 0.25 0.75 0.74 0.65 https://doi.org/10.1371/journal.pcbi.1011055.t001 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1011055 April 24, 2023 10 / 20 Table 1. Comparison of aorta dimensions between original real cohort (n = 67) and synthetic cohort (n = 3000). 10 / 20 PLOS COMPUTATIONAL BIOLOGY Validation with real patient data Coefficients for the first 10 shape modes for all new subjects (n = 10) were found to lie within the max-min ranges of the original PCA shape modes (n = 67, see S6 Fig). The average prediction error using the MNAES metric (ML vs CFD computed on SSM meshes) was Computational Biology \| https://doi.org/10.1371/journal.pcbi.1011055 April 24, 2023 11 / 20 PLOS COMPUTATIONAL BIOLOGY Deep neural networks for fast aortic 3D pressure and velocity flow fields Fig 9. Best, median and worst test-set predictions. Comparisons between ground truth (CFD) and predicted (ML) in the test set (n = 200). Best, median and worst cases for both pressure and velocity-magnitude are shown, ranked using the mean node-to-node error (MNAES). Pressure/velocity gradients are also displayed (black lines = CFD, red = ML). htt //d i /10 1371/j l bi 1011055 009 ns between ground truth (CFD) and predicted (ML) in the test set (n = 200). Best, median and worst d using the mean node-to-node error (MNAES). Pressure/velocity gradients are also displayed (black Fig 9. Best, median and worst test-set predictions. Comparisons between ground truth (CFD) and predicted (ML) in the test set (n = 200). Best, median and worst cases for both pressure and velocity-magnitude are shown, ranked using the mean node-to-node error (MNAES). Pressure/velocity gradients are also displayed (black lines = CFD, red = ML). https://doi.org/10.1371/journal.pcbi.1011055.g009 10.19% ±10.41 and 4.47% ±1.18 for pressure and velocity respectively. This corresponds to an increase in mean subject error by 4.18% for pressure and 0.48% for velocity, when compared to the population error in the test-set of synthetic cases (n = 200). Due to the lack of point correspondence and surface matching between the real and SSM shapes, Frechet distance of pressure and velocity gradients were used to compare the ML CFD results with the patient CFD data (computed using both SSM and real geometries). The FD computed between the ML and the CFD (SSM) gradients corresponded to the prediction error arising solely from the ML model (FD SSM). The FD computed between the ML and the CFD (real shape) gradients corresponds to the total error between the ML prediction and true CFD Computational Biology \| https://doi.org/10.1371/journal.pcbi.1011055 April 24, 2023 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1011055 April 24, 2023 12 / 20 PLOS COMPUTATIONAL BIOLOGY Deep neural networks for fast aortic 3D pressure and velocity flow fields Fig 10. Shape modes vs. ML error. Validation with real patient data Scatter plot comparing the shape PCA mode values against subject error (MNAES) in the test set (n = 200). Pearson R coefficients and p-values were computed for each subplot. https://doi.org/10.1371/journal.pcbi.1011055.g010 Fig 10. Shape modes vs. ML error. Scatter plot comparing the shape PCA mode values against subject error (MNAES) in the test set (n = 200). Pearson R coefficients and p-values were computed for each subplot. https://doi.org/10.1371/journal.pcbi.1011055.g010 https://doi.org/10.1371/journal.pcbi.1011055.g010 https://doi.org/10.1371/journal.pcbi.1011055.g010 (FD real). The subjects with the best and worst FD (real) for pressure and velocity are shown in Fig 11. The mean FD SSM was 1680 ±629 Pa for pressure and 0.47 ±0.17 m/s for velocity. This compares to 4583 ±3210 Pa for pressure and 1.30 ±0.37 m/s for velocity between the ML and real-CFD (FD real). (FD real). The subjects with the best and worst FD (real) for pressure and velocity are shown in Fig 11. The mean FD SSM was 1680 ±629 Pa for pressure and 0.47 ±0.17 m/s for velocity. This compares to 4583 ±3210 Pa for pressure and 1.30 ±0.37 m/s for velocity between the ML and real-CFD (FD real). PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1011055 April 24, 2023 Model performance Our ML models were observed to predict point clouds of both pressure and velocity flow fields with good accuracy, while being approximately 4,000x faster than our conventional CFD method. Node errors for pressure were seen to be larger at the inlet, while velocity errors were more skewed towards the distal regions of the aorta (Fig 8, left). This is most likely due to the CFD boundary conditions at the inlet and outlet constraining the pressure/velocity variability at these regions. The further away from the aortic inlet or outlet, the greater the variability in velocity or pressure, respectively. It should be noted that there were no significant biases in either pressure or velocity predictions, suggesting that there were no systematic errors with the models (Fig 8, right). In testing, we found that the mean pressure subject error was slightly higher than the mean velocity error. This is despite the pressure PCA model capturing more variance than the velocity model. A possible explanation for the higher pressure errors is that the association between shape and pressure is more complex than that between shape and velocity in aortic domains. This is supported by the observation that shape modes do not cor- relate with pressure errors (Fig 10). Interestingly, there was a strong negative correlation between shape mode 3 and velocity error. This suggests that more ‘gothic’ aortas (characterised by a more triangular arch) were less prone to velocity prediction errors. A possible explanation may be that the gothic arch constrains downstream flow patterns (where most velocity errors occur), hence making it easier for the model to characterise flow features associated to this sub- set of aortic shapes. An important element of this study was to apply trained DNN models to prospective, unseen cases in order to explore the feasibility of performing inference on real patient data (n = 10). It was shown that the average node-based prediction error (MNAES) on the prospec- tive cases did increase for pressure from 6.01% to 10.19%. Velocity errors increased only mar- ginally in the prospective test cases (3.99% to 4.47%). This aligns with the previous observation that the relationship between shape and pressure may be more complex due to the lack of an observed correlation between the shape PCA modes and pressure prediction errors. We also compared the ML predictions to the real CFD (performed on the raw segmentation mesh) for all cases. Discussion The main findings of this study were: i) statistical shape models (SSMs) and PCA are suitable for creating synthetic training data and dimensionality-reduced representations of 3D shape and flow, ii) DNNs based on these dimensionality-reduced representations can predict pres- sure and velocity fields with high accuracy. Fig 11. Testing on prospective data. Best and worst pressure and velocity predictions on the real patient test cohort (n = 10). FD (SSM) is the error between the predicted (red) and SSM CFD (dashed black) gradients. FD (real) is the error between the predicted (red) and true CFD (solid black) gradients. https://doi org/10 1371/journal pcbi 1011055 g011 Fig 11. Testing on prospective data. Best and worst pressure and velocity predictions on the real patient test cohort (n = 10). FD (SSM) is the error between the predicted (red) and SSM CFD (dashed black) gradients. FD (real) is the error between the predicted (red) and true CFD (solid black) gradients. Fig 11. Testing on prospective data. Best and worst pressure and velocity predictions on the real patient test cohort (n = 10). FD (SSM) is the error between the predicted (red) and SSM CFD (dashed black) gradients. FD (real) is the error between the predicted (red) and true CFD (solid black) gradients. https://doi org/10 1371/journal pcbi 1011055 g011 Fig 11. Testing on prospective data. Best and worst pressure and velocity predictions on the real patient test cohort (n = 10). FD (SSM) is the error between the predicted (red) and SSM CFD (dashed black) gradients. FD (real) is the error between the predicted (red) and true CFD (solid black) gradients. https://doi.org/10.1371/journal.pcbi.1011055.g011 https://doi.org/10.1371/journal.pcbi.1011055.g011 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1011055 April 24, 2023 13 / 20 PLOS COMPUTATIONAL BIOLOGY Deep neural networks for fast aortic 3D pressure and velocity flow fields Synthetic data generation and dimensionality reduction A key element of our approach was to use an SSM and PCA to both generate a synthetic train- ing dataset (n = 3,000), and to parameterise aortic shape/flow fields for simplifying DNN model training. We demonstrated that volumetric meshes generated by our SSM were suitable for forcing point correspondence in our dataset through interpolation of 3D aortic CFD flow fields. It was also shown that less than 60 PCA modes were needed to capture the majority of variance within aortic shape deformations and 3D pressure/velocity fields. Mean PCA recon- struction errors of pressure/velocity were found to be low, but not insignificant in the worst observed test-set reconstructions (S3 Fig). Geometric properties of our synthetic data were shown to be mostly close to the real cohort (Table 1). In the future, newer approaches for gen- erating synthetic data and creating dimensionality-reduced representations of complex struc- tures could be explored, notably deep-learning methods such as autoencoders [11] and generative adversarial networks (GANs) [26]. In some studies, autoencoders have been shown to be superior to PCA-based methods (e.g. for 3D facial surface reconstruction) [27, 28]. Addi- tionally, GANs have shown promise for creating images of synthetic patients afflicted with CHD [29]. Finally, instead of sampling a Gaussian distribution to find combinations of shape PCA parameters, other methods for creating new DNN training data may be more appropri- ate, such as Latin hypercube sampling [30]. Limitations In order to translate our DNN-based CFD approach to clinics, there are two main modelling limitations which need to be bypassed. The first is related to the loss in surface accuracy when using SSM representations of aortic shapes. The second is the current simplicity of the CFD approach, which needs to be further developed in order to generate more meaningful DNN training data. Model performance This enabled the proportion of the total error due to the SSM and DNN to be PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1011055 April 24, 2023 14 / 20 PLOS COMPUTATIONAL BIOLOGY Deep neural networks for fast aortic 3D pressure and velocity flow fields estimated. We showed *60% of the total gradient error in pressure/velocity was due to the SSM, which further strengthens the argument for improved shape parameterisation. The approach of using DNNs to model 3D aortic pressure and velocity flow fields has been described in other works [10, 11]. However, an important limitation of this data-driven approach is that flow fields cannot be assumed to satisfy the Navier-Stokes equations for incompressible flow. This may hinder the applicability of the approach for simulations where accurate flow field prediction is insufficient in of itself, and conservation of mass and momen- tum needs to be guaranteed. Future development should also include models for computing velocity x, y and z components, allowing visualisation of streamlines or possible derivation of parameters such as wall-shear stress. Although we used fully-connected DNNs, other studies have reported the use of long short-term memory (LSTM) networks or specialised architec- tures such as PointNet to build CFD-based ML models [9, 31, 32]. LSTM networks in particu- lar may be highly suited towards any ML transient flow applications, due to their inherent ability to learn temporal sequences of data. Other architectures such as probabilistic DNNs which output uncertainty intervals during inference should also be explored [31, 33, 34]. Addi- tionally, alternative methods for sampling synthetic data (e.g. Latin hypercube sampling) may produce a more diverse dataset for model training. This may prevent the occurrence of outli- ers, such as the worst pressure case in the test-set (MNAES=23.6%). Potential clinical utility We believe that the fast computation of haemodynamics using our method has multiple clini- cal uses. However, this is a proof-of-concept study and further improvements are required prior to any clinical validation (particularly the inclusion of patient-specific boundary condi- tions and time varying flow fields—see limitations). Nevertheless, if this could be achieved we envisage several clinical uses, such as for supporting the identification of patients who need an intervention and predicting the outcome. Specific to our population, several studies have shown that CFD can be used to evaluate abnormal haemodynamics (particularly during stress) and predict normalisation of haemodynamics after stenting of coarctation. However, this approach is rarely used in the clinical environment because it is so time consuming. We believe our approach could be extrapolated to evaluate stress haemodynamics by simulating each training dataset case under elevated cardiac stress conditions. A second application could involve a fast and automatic pipeline to predict post-stenting haemodynamics in aortas. This could be implemented in a two-step solution using: (i) a surrogate finite-element model for predicting an ideal post-op aortic shape following stenting, and (ii) a surrogate CFD model for predicting haemodynamics on the post-op aortic shape (following the approach we present in this study). Use of such models would bring forth a new level of precision medicine that is cur- rently lacking in congenital heart disease. Shape parameterisation It has been seen in previous studies that aortic CFD flow fields are highly sensitive to geometric and topological variation [35, 36]. For this reason, using shape vectors that are accurate 15 / 20 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1011055 April 24, 2023 PLOS COMPUTATIONAL BIOLOGY Deep neural networks for fast aortic 3D pressure and velocity flow fields descriptors of the aortic surfaces is of critical importance. In the future, the relationship between the SSM registration error and the resultant CFD flow fields should be further investi- gated. Where possible, augmentations to the shape vector should be trialled in order to see if DNN prediction errors for real subjects can be reduced. This may require including additional shape information as inputs in the DNN models to act as a form of regularisation, such as a registration error or a geometric feature (e.g. centreline diameters). Such complimentary shape descriptors may be used to better inform the network of important features not fully captured by the SSM shape vector alone. Additionally, multiple SSMs based upon templates other than the mean aortic shape could be generated. These would enable closer non-rigid reg- istration for unique cases where the target aorta deviates significantly from the mean shape. Recently, Wiputra et al. demonstrated methods for augmenting SSMs to enable the inclusion of head and neck vessel geometry within the aortic shape parameterisation, while retaining high accuracy with low-dimensional PCA vectors [37]. A similar approach may be employed in future studies in order to be able to fully describe the patient-specific aorta with head and neck vessels. Of course, a simple initial improvement could be made by adding more subjects to our SSM to introduce more variability in the population. Computational fluid dynamics In this study, a simplified CFD pipeline was chosen in order to easily automate and ensure convergence for numerous simulations (n = 3,000). A standard CFD solver set-up was used, assuming steady-state conditions and incompressible flow. In order to account for the pulsati- lity involved in aortic flow, a transient solver may be better suited to real-life applications. It was also assumed that the flow through the great arteries at peak systole was laminar [38]. In the future, the Reynolds number may be computed for individual cases to allow for the inclu- sion of turbulence modelling where necessary. However, for complex morphologies (such as CoA), Reynolds number has been seen to be an inconsistent measure of turbulence [39]. Feiger et al. proposed an alternative solution to turbulence modelling when performing CFD on a large scale, which involves meshing the domain with extremely high numbers of nodes [10]. Boundary conditions selected included a fixed, flat velocity inlet and zero pressure outlet condition for all cases. However, idealised inlet velocity profiles (flat, parabolic etc.) have been shown to be ineffective for producing clinically relevant data [40, 41]. Thamsen et al. showed that a synthetic aortic population could be created with realistic accompanying 4D MRI- derived vector flow profiles [42]. In the future, a similar approach could be taken, allowing for an additional velocity vector field input parameter into the ML model. Alternatively, a more accessible approach could involve the use of a parabolic velocity inlet condition and a patient- specific unsteady flow profile (derived from phase-contrast MRI) in conjunction with a tran- sient solver for resolving the peak systolic flow field. In this study, it was decided to omit the head and neck vessels from the CFD model for simplicity, however this would be required when aiming to simulate realistic patient-specific aortic haemodynamics [36]. Indeed, Wiputra et al. showed that accurate modelling of the head and neck vessels is necessary for capturing local flow features in the arch and producing realistic downstream fluid flow forces [37]. Thus, the inclusion of head and neck vessels along with lumped parameter outlet models such as Windkessel models should be explored in the future [43, 44], as modelling downstream resis- tance has been seen to produce more clinically meaningful results [45–47]. Supporting information S1 Fig. Volume mesh deformation: Mesh skewness. An example of template volume mesh deformation when generating the volume mesh for a new subject is shown. (EPS) S2 Fig. CFD sensitivity analysis. A test aortic shape with a sharp arch angulation was chosen to be used to perform a mesh sensitivity study. (EPS) S2 Fig. CFD sensitivity analysis. A test aortic shape with a sharp arch angulation was chosen to be used to perform a mesh sensitivity study. (EPS) S3 Fig. Best and worst PCA errors. PCA reconstructions in the test cohort (n = 200) were performed to assess the level of information loss due to dimensionality reduction. (EPS) S4 Fig. The final pressure and velocity DNN architectures. (EPS) S4 Fig. The final pressure and velocity DNN architectures. (EPS) S5 Fig. Regional Bland-Altman analysis. This was conducted on three different regions of the aorta (ascending, transverse arch, descending). (EPS) S5 Fig. Regional Bland-Altman analysis. This was conducted on three different regions of the aorta (ascending, transverse arch, descending). (EPS) S6 Fig. PCA projections of real cases. All ten new cases had their first 35 shape mode scores plotted against the range of the original dataset scores. (EPS) S6 Fig. PCA projections of real cases. All ten new cases had their first 35 shape mode scores plotted against the range of the original dataset scores. (EPS) Conclusion In this proof-of-concept study, we have proposed a pipeline for building ML-based models to perform repetitive vessel-based CFD tasks. Generation of synthetic aortic training data by 16 / 20 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1011055 April 24, 2023 PLOS COMPUTATIONAL BIOLOGY Deep neural networks for fast aortic 3D pressure and velocity flow fields means of shape modelling allowed ML techniques to be used, even where data scarcity is an issue (n = 67). Point correspondence was maintained between subject meshes in order to enable PCA. Our ML models were able to compute pressure and velocity flow fields much more rapidly (4,000x) than traditional CFD solvers, without large computational requirements or simulation setup. Comparison between predicted and ground truth test cases revealed good overall performance. Testing on prospective cases revealed that shape registration errors could produce misleading flow fields which deviated significantly from the ‘real’ CFD result, even in the presence of low ML errors. The approach described in this study is shape-driven and is applicable to any vascular structure which can be segmented from medical images. In the future, the models should be improved, so they can perform inference on prospective data from real patients. The requirements for this are two-fold; improving the accuracy of the shape representation methods while incorporating the head and neck vessels, and using a more real- istic CFD pipeline for generating training data with the inclusion of patient-specific boundary conditions. Comparison of the ML models against clinically acquired data (such as catheter- based pressure drops) should be also performed in the future for validation purposes. References 1. Morris PD, Narracott A, Von Tengg-Kobligk H, Soto DAS, Hsiao S, Lungu A, et al. Computational fluid dynamics modelling in cardiovascular medicine. Heart. 2016; 102(1):18–28. https://doi.org/10.1136/ heartjnl-2015-308044 PMID: 26512019 2. Biglino G, Cosentino D, Steeden JA, De Nova L, Castelli M, Ntsinjana H, et al. Using 4D Cardiovascular Magnetic Resonance Imaging to Validate Computational Fluid Dynamics: A Case Study. Frontiers in Pediatrics. 2015; 3(December):1–10. https://doi.org/10.3389/fped.2015.00107 PMID: 26697416 3. Zhu Y, Chen R, Juan YH, Li H, Wang J, Yu Z, et al. Clinical validation and assessment of aortic hemody- namics using computational fluid dynamics simulations from computed tomography angiography. Bio- Medical Engineering Online. 2018; 17(1):1–12. https://doi.org/10.1186/s12938-018-0485-5 PMID: 29720173 4. LaDisa JF, Alberto Figueroa C, Vignon-Clementel IE, Jin Kim H, Xiao N, Ellwein LM, et al. Computa- tional simulations for aortic coarctation: representative results from a sampling of patients. Journal of Biomedical Engineering. 2011. https://doi.org/10.1115/1.4004996 PMID: 22010743 5. Itu L, Sharma P, Ralovich K, Mihalef V, Ionasec R, Everett A, et al. Non-invasive hemodynamic assess- ment of aortic coarctation: validation with in vivo measurements. Annals of biomedical engineering. 2013; 41(4):669–81. https://doi.org/10.1007/s10439-012-0715-0 PMID: 23232558 6. Qiu Y, Wang J, Zhao J, Wang T, Zheng T, Yuan D. Association between blood flow pattern and rupture risk of abdominal aortic aneurysm based on computational fluid dynamics. European Journal of Vascu- lar and Endovascular Surgery. 2022; 64(2–3):155–64. https://doi.org/10.1016/j.ejvs.2022.05.027 PMID: 35605907 7. Huberts W, Heinen SGH, Zonnebeld N, van den Heuvel DAF, de Vries JPPM, Tordoir JHM, et al. What is needed to make cardiovascular models suitable for clinical decision support? A viewpoint paper. Jour- nal of Computational Science. 2018; 24:68–84. Available from: https://doi.org/10.1016/j.jocs.2017.07. 006 8. Avendi MR, Kheradvar A, Jafarkhani H. A combined deep-learning and deformable-model approach to fully automatic segmentation of the left ventricle in cardiac MRI. Medical image analysis. 2016; 30:108– 19. https://doi.org/10.1016/j.media.2016.01.005 PMID: 26917105 9. Yevtushenko P, Goubergrits L, Gundelwein L, Setio A, Heimann T, Ramm H, et al. Deep Learning Based Centerline-Aggregated Aortic Hemodynamics: An Efficient Alternative to Numerical Modelling of Hemodynamics. IEEE Journal of Biomedical and Health Informatics. 2021. 10. Feiger B, Gounley J, Adler D, Leopold JA, Draeger EW, Chaudhury R, et al. Accelerating massively par- allel hemodynamic models of coarctation of the aorta using neural networks. Scientific Reports. 2020; 10(1):1–13. Available from: https://doi.org/10.1038/s41598-020-66225-0 PMID: 32528104 11. Liang L, Mao W, Sun W. A feasibility study of deep learning for predicting hemodynamics of human tho- racic aorta. Author Contributions Conceptualization: Endrit Pajaziti, Javier Montalt-Tordera, Claudio Capelli, Raphae¨l Sivera, Emilie Sauvage, Michael Quail, Silvia Schievano, Vivek Muthurangu. Conceptualization: Endrit Pajaziti, Javier Montalt-Tordera, Claudio Capelli, Raphae¨l Sivera, Emilie Sauvage, Michael Quail, Silvia Schievano, Vivek Muthurangu. Data curation: Michael Quail. Formal analysis: Endrit Pajaziti. Investigation: Endrit Pajaziti. Methodology: Endrit Pajaziti, Javier Montalt-Tordera, Claudio Capelli, Raphae¨l Sivera, Silvia Schievano, Vivek Muthurangu. Data curation: Michael Quail. Formal analysis: Endrit Pajaziti. Methodology: Endrit Pajaziti, Javier Montalt-Tordera, Claudio Capelli, Raphae¨l Sivera, Silvia Schievano, Vivek Muthurangu. 17 / 20 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1011055 April 24, 2023 PLOS COMPUTATIONAL BIOLOGY Deep neural networks for fast aortic 3D pressure and velocity flow fields Software: Endrit Pajaziti, Raphae¨l Sivera. Supervision: Claudio Capelli, Emilie Sauvage, Silvia Schievano, Vivek Muthurangu. Writing – original draft: Endrit Pajaziti. Writing – review & editing: Endrit Pajaziti, Javier Montalt-Tordera, Claudio Capelli, Raphae¨l Sivera, Emilie Sauvage, Michael Quail, Silvia Schievano, Vivek Muthurangu. Software: Endrit Pajaziti, Raphae¨l Sivera. Writing – review & editing: Endrit Pajaziti, Javier Montalt-Tordera, Claudio Capelli, Raphae¨l Sivera, Emilie Sauvage, Michael Quail, Silvia Schievano, Vivek Muthurangu. References Journal of Biomechanics. 2020; 99. Available from: https://doi.org/10.1016/j.jbiomech.2019. 109544 PMID: 31806261 12. Hoeijmakers MJMM, Waechter-Stehle I, Weese J, Van de Vosse FN. Combining statistical shape modeling, CFD, and meta-modeling to approximate the patient-specific pressure-drop across the aortic valve in real-time. International Journal for Numerical Methods in Biomedical Engineering. 2020; 36 (10):1–18. https://doi.org/10.1002/cnm.3387 PMID: 32686898 13. Lee SW, Antiga L, Spence JD, Steinman DA. Geometry of the carotid bifurcation predicts its exposure to disturbed flow. Stroke. 2008; 39(8):2341–7. https://doi.org/10.1161/STROKEAHA.107.510644 PMID: 18556585 14. Antiga L, Piccinelli M, Botti L, Ene-Iordache B, Remuzzi A, Steinman DA. An image-based modeling framework for patient-specific computational hemodynamics. Medical & biological engineering & com- puting. 2008; 46(11):1097–112. https://doi.org/10.1007/s11517-008-0420-1 PMID: 19002516 15. Besl PJ, McKay ND. Method for registration of 3-D shapes. In: Sensor fusion IV: control paradigms and data structures. vol. 1611. Spie; 1992. p. 586–606. 18 / 20 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1011055 April 24, 2023 PLOS COMPUTATIONAL BIOLOGY Deep neural networks for fast aortic 3D pressure and velocity flow fields 16. Bruse JL, McLeod K, Biglino G, Ntsinjana HN, Capelli C, Hsia TY, et al. A statistical shape modelling framework to extract 3D shape biomarkers from medical imaging data: assessing arch morphology of repaired coarctation of the aorta. BMC medical imaging. 2016; 16(1):1–19. https://doi.org/10.1186/ s12880-016-0142-z PMID: 27245048 17. Boˆne A, Louis M, Martin B, Durrleman S. Deformetrica 4: an open-source software for statistical shape analysis. In: International Workshop on Shape in Medical Imaging. Springer; 2018. p. 3–13. 18. Piccinelli M, Veneziani A, Steinman DA, Remuzzi A, Antiga L. A framework for geometric analysis of vascular structures: application to cerebral aneurysms. IEEE transactions on medical imaging. 2009; 28 (8):1141–55. https://doi.org/10.1109/TMI.2009.2021652 PMID: 19447701 19. Hang S. TetGen, a Delaunay-based quality tetrahedral mesh generator. ACM Trans Math Softw. 2015; 41(2):11. 20. Garcia J, van der Palen RL, Bollache E, Jarvis K, Rose MJ, Barker AJ, et al. Distribution of blood flow velocity in the normal aorta: effect of age and gender. Journal of Magnetic Resonance Imaging. 2018; 47(2):487–98. https://doi.org/10.1002/jmri.25773 PMID: 28556277 21. Powell A, Maier S, Chung T, Geva T. Phase-velocity cine magnetic resonance imaging measurement of pulsatile blood flow in children and young adults: in vitro and in vivo validation. Pediatric cardiology. 2000; 21(2):104–10. https://doi.org/10.1007/s002469910014 PMID: 10754076 22. Bonfanti M, Balabani S, Greenwood JP, Puppala S, Homer-Vanniasinkam S, Dı´az-Zuccarini V. References Compu- tational tools for clinical support: a multi-scale compliant model for haemodynamic simulations in an aor- tic dissection based on multi-modal imaging data. Journal of The Royal Society Interface. 2017; 14 (136):20170632. https://doi.org/10.1098/rsif.2017.0632 PMID: 29118115 23. Kingma DP, Ba J. Adam: A method for stochastic optimization. arXiv preprint arXiv:14126980. 2014. 24. Bergstra J, Bardenet R, Bengio Y, Ke´gl B. Algorithms for hyper-parameter optimization. Advances in neural information processing systems. 2011; 24. 25. Eiter T, Mannila H. Computing discrete Fre´chet distance. Technical Report CD-TR 94/64. 1994. 26. Creswell A, White T, Dumoulin V, Arulkumaran K, Sengupta B, Bharath AA. Generative adversarial net- works: An overview. IEEE signal processing magazine. 2018; 35(1):53–65. https://doi.org/10.1109/ MSP.2017.2765202 27. Ranjan A, Bolkart T, Sanyal S, Black MJ. Generating 3D faces using convolutional mesh autoencoders. In: Proceedings of the European Conference on Computer Vision (ECCV); 2018. p. 704–20. 28. Wang Y, Yao H, Zhao S. Auto-encoder based dimensionality reduction. Neurocomputing. 2016; 184:232–42. https://doi.org/10.1016/j.neucom.2015.08.104 29. Diller GP, Vahle J, Radke R, Vidal MLB, Fischer AJ, Bauer UM, et al. Utility of deep learning networks for the generation of artificial cardiac magnetic resonance images in congenital heart disease. BMC Medical Imaging. 2020; 20(1):1–8. https://doi.org/10.1186/s12880-020-00511-1 PMID: 33032536 30. Stein M. Large sample properties of simulations using Latin hypercube sampling. Technometrics. 1987; 29(2):143–51. https://doi.org/10.1080/00401706.1987.10488205 31. Abbas A, Rafiee A, Haase M, Malcolm A. Geometric Convolutional Neural Networks–A Journey to Sur- rogate Modelling of Maritime CFD. In: The 9th Conference on Computational Methods in Marine Engi- neering (Marine 2021); 2022. p. 1–8. 32. Qi CR, Su H, Mo K, Guibas LJ. PointNet: Deep Learning on Point Sets for 3D Classification and Seg- mentation. CVPR 2017 Open Access. 2016. Available from: https://arxiv.org/abs/1612.00593 33. Umetani N, Bickel B. Learning Three-Dimensional Flow for Interactive Aerodynamic Design regression prediction for new shape. ACM Trans Graph. 2018; 37(4). 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Computer meth- ods in biomechanics and biomedical engineering. 2022:1–15. https://doi.org/10.1080/10255842.2022. 2128672 PMID: 36165506 19 / 20 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1011055 April 24, 2023 PLOS COMPUTATIONAL BIOLOGY Deep neural networks for fast aortic 3D pressure and velocity flow fields 38. Caballero AD, Laı´n S. A review on computational fluid dynamics modelling in human thoracic aorta. Car- diovascular Engineering and Technology. 2013; 4(2):103–30. https://doi.org/10.1007/s13239-013- 0146-6 39. Lantz J, Ebbers T, Engvall J, Karlsson M. Numerical and experimental assessment of turbulent kinetic energy in an aortic coarctation. Journal of biomechanics. 2013; 46(11):1851–8. https://doi.org/10.1016/ j.jbiomech.2013.04.028 PMID: 23746596 40. Youssefi P, Gomez A, Arthurs C, Sharma R, Jahangiri M, Alberto Figueroa C. Impact of patient-specific inflow velocity profile on hemodynamics of the thoracic aorta. 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https://openalex.org/W4231092279	https://www.qeios.com/read/A16RA8/pdf	English	null	Eyelid	Definitions	2,020	cc-by	65	Eyelid National Cancer Institute Qeios ID: A16RA8 · https://doi.org/10.32388/A16RA8 Qeios · Definition, February 2, 2020 Open Peer Review on Qeios Open Peer Review on Qeios Open Peer Review on Qeios Eyelid Open Peer Review on Qeios Source National Cancer Institute. Eyelid. NCI Thesaurus. Code C12713. A thin membrane of skin with the purpose of covering and protecting an eye. Qeios ID: A16RA8 · https://doi.org/10.32388/A16RA8 1/1
https://openalex.org/W2888043750	https://europepmc.org/articles/pmc6153855?pdf=render	English	null	Correction to: Monitoring Quality Across Home Visiting Models: A Field Test of Michigan’s Home Visiting Quality Assurance System	Maternal and child health journal	2,018	cc-by	462	Correction to: Monitoring Quality Across Home Visiting Models: A Field Test of Michigan’s Home Visiting Quality Assurance System Julia Heany1 · Jennifer Torres1 · Cynthia Zagar2 · Tiffany Kostelec3 Published online: 22 August 2018 © The Author(s) 2018 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 use, duplication, adapta- tion, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made. Maternal and Child Health Journal (2018) 22 (Suppl 1):S116 https://doi.org/10.1007/s10995-018-2617-8 Maternal and Child Health Journal (2018) 22 (Suppl 1):S116 https://doi.org/10.1007/s10995-018-2617-8 CORRECTION Correction to: Maternal and Child Health Journal https://doi.org/10.1007/s10995-018-2538-6 distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made. The article “Monitoring Quality Across Home Visiting Models: A Field Test of Michigan’s Home Visiting Qual- ity Assurance System”, written by Julia Heany, Jennifer Torres, Cynthia Zagar and Tiffany Kostelec, was originally published electronically on the publisher’s internet portal (currently SpringerLink) on 05 June 2018 without open access. With the author(s)’ decision to opt for Open Choice the copyright of the article changed on 20 July 2018 to © The Author(s) 2018 and the article is forthwith distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licen ses/by/4.0/), which permits use, duplication, adaptation, The original article has been corrected. 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 use, duplication, adapta- tion, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made. 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 use, duplication, adapta- tion, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made. The original article can be found online at https://doi.org/10.1007/ s10995-018-2538-6. * Julia Heany jheany@mphi.org * Julia Heany jheany@mphi.org 1 Michigan Public Health Institute, Center for Healthy Communities, 2342 Woodlake Drive, Okemos, MI 48864, USA 2 Michigan Public Health Institute, Public Health Services, 2364 Woodlake Drive, Suite 180, Okemos, MI 48864, USA 3 Michigan Department of Health and Human Services, 109 W. Michigan Ave, 7th FL, Lansing, MI 48933, USA :.(123456789 3
https://openalex.org/W2949447891	https://figshare.com/articles/journal_contribution/Physical_activity_of_children_and_adolescents_with_Charcot-Marie-Tooth_neuropathies_A_cross-sectional_case-controlled_study/8264372/1/files/15452789.pdf	English	null	Physical activity of children and adolescents with Charcot-Marie-Tooth neuropathies: A cross-sectional case-controlled study	PloS one	2,019	cc-by	1,999	Physical activity in paediatric CMT Physical activity in paediatric CMT Supplementary material – Physical Activity Questionnaire – child (PAQ-C) adapted for Australian participants Supplementary material – Physical Activity Questionnaire – child (PAQ-C) adapted for Australian participants Instructions • We would like to know about your level of physical activity from the last 7 days (the last week). • This includes sports or dance that make you sweat or make your legs feel tired, or games that make you breathe hard, like playing chasey, skipping, running, climbing and others. • There are no right or wrong answers – this is not a test. • Please answer all the questions as honestly and accurately as you can – this is important. • If your parent or another adult helped you fill in this questionnaire please tick the box “I had help ☺”. • If you have any concerns about these questions or how they make you feel, please let your parent and / or the physiotherapist who is doing your assessment know. Study ID: Date: I had help ☺ • We would like to know about your level of physical activity from the last 7 days (the last week). In the last 7 days, what did you do most of the time at recess / little play? (Tick one only) Sat down (talking, reading, doing schoolwork) Stood around or walked around Ran or played a little bit Ran around and played quite a bit Ran and played hard most of the time 4. In the last 7 days, what did you normally do at lunch (besides eating lunch)? (Tick one only) Sat down (talking, reading, doing schoolwork) Stood around or walked around Ran or played a little bit Ran around and played quite a bit Ran and played hard most of the time 5. In the last 7 days, on how many days right after school did you do sports, dance or play games in which you were very active? This can include Out of school hours care Physical activity in paediatric CMT Basketball / netball Other 2. In the last 7 days, during your sports and / or physical education (PE) classes, how often were you very active (playing hard, running, jumping, throwing)? (Tick one only) I don’t do PE Hardly ever Sometimes Quite often Always 3. In the last 7 days, what did you do most of the time at recess / little play? (Tick one only) Sat down (talking, reading, doing schoolwork) Stood around or walked around Ran or played a little bit Ran around and played quite a bit Ran and played hard most of the time 4. In the last 7 days, what did you normally do at lunch (besides eating lunch)? (Tick one only) Sat down (talking, reading, doing schoolwork) Stood around or walked around Ran or played a little bit Ran around and played quite a bit Ran and played hard most of the time 5. In the last 7 days, on how many days right after school did you do sports, dance or play games in which you were very active? This can include Out of school hours care (OHSC) (Tick one only) None 1 time last week 2 or 3 times last week 2. In the last 7 days, during your sports and / or physical education (PE) classes, how often were you very active (playing hard, running, jumping, throwing)? (Tick one only) I don’t do PE Hardly ever Sometimes Quite often 4. In the last 7 days, what did you normally do at lunch (besides eating lunch)? • We would like to know about your level of physical activity from the last 7 days (the last week). • We would like to know about your level of physical activity from the last 7 days (the last week). (the last week). • This includes sports or dance that make you sweat or make your legs feel tired, or games that make you breathe hard, like playing chasey, skipping, running, climbing and others. • There are no right or wrong answers – this is not a test. • Please answer all the questions as honestly and accurately as you can – this is important. • If your parent or another adult helped you fill in this questionnaire please tick the box “I had help ☺”. • If you have any concerns about these questions or how they make you feel, please let your parent and / or the physiotherapist who is doing your assessment know. Study ID: Date: I had help ☺ • This includes sports or dance that make you sweat or make your legs feel tired, or games that make you breathe hard, like playing chasey, skipping, running, climbing and others. • There are no right or wrong answers – this is not a test. • Please answer all the questions as honestly and accurately as you can – this is important. 1. Physical activity in your spare time (time not in school or in organised sports / classes): Have you done any of the following activities in the past 7 days (last one week)? If yes, how many times? Tick one box per row. 1. Physical activity in your spare time (time not in school or in organised sports / classes): Have you done any of the following activities in the past 7 days (last one week)? If yes, how many times? Tick one box per row. No 1-2 3-4 5-6 7 times or more. Skipping Water sports Roller blading/skating Chasey / tag Walking for exercise Walking to/from school or public transport Bicycling for fun / recreation Bicycling to/from school Jogging or running Aerobics / gym Swimming for fun/recreation Cricket / baseball / softball Dance Football Soccer Skateboarding Hockey Physical activity in paediatric CMT Basketball / netball Other 2. In the last 7 days, during your sports and / or physical education (PE) classes, how often were you very active (playing hard, running, jumping, throwing)? (Tick one only) I don’t do PE Hardly ever Sometimes Quite often Always 3. • We would like to know about your level of physical activity from the last 7 days (the last week). (Tick one only) Sat down (talking, reading, doing schoolwork) Stood around or walked around Ran or played a little bit Ran around and played quite a bit Ran and played hard most of the time 4. In the last 7 days, what did you normally do at lunch (besides eating lunch)? (Tick one only) Sat down (talking, reading, doing schoolwork) Stood around or walked around Ran or played a little bit Ran around and played quite a bit Ran and played hard most of the time 5. In the last 7 days, on how many days right after school did you do sports, dance or play games in which you were very active? This can include Out of school hours care (OHSC) (Tick one only) None 1 time last week 2 or 3 times last week 5. In the last 7 days, on how many days right after school did you do sports, dance or play games in which you were very active? This can include Out of school hours care (OHSC) (Tick one only) None 1 time last week 2 or 3 times last week Physical activity in paediatric CMT 4 times last week 5 times last week 6. In the last 7 days, on how many evenings did you do sports, dance or play games in which you were very active? (Tick one only) None 1 time last week 2 or 3 times last week 4-5 times last week 6-7 times last week 6. In the last 7 days, on how many evenings did you do sports, dance or play games which you were very active? (Tick one only) 6. In the last 7 days, on how many evenings did you do sports, dance or play games in which you were very active? (Tick one only) None 1 time last week 2 or 3 times last week 4-5 times last week 6-7 times last week 7. On the last week-end, how many times did you do sports, dance or play games in which you were very active? (Tick one only) None 1 time 2 or 3 times 4-5times 6 or more times 7. On the last week-end, how many times did you do sports, dance or play games in which you were very active? (Tick one only) None 1 time 2 or 3 times 4-5times 6 or more times 7. • We would like to know about your level of physical activity from the last 7 days (the last week). On the last week-end, how many times did you do sports, dance or play games in which you were very active? (Tick one only) 8. Which one of the following describes you best for the last 7 days? Read all five statements before deciding on the one answer that describes you. (Tick one only) A. All or most of my free time was spend doing things that involve little physical effort B. I sometimes (1-2 times last week) did physical things in my free time (eg. Played sports, went running, swimming, bike riding etc) C. I often (3-4 times last week) did physical things in my free time D. I quite often (5-6 times last week) did physical things in my free time E. I very often (7 or more times last week) did physical things in my free time 8. Which one of the following describes you best for the last 7 days? Read all five statements before deciding on the one answer that describes you. (Tick one only) A. All or most of my free time was spend doing things that involve little physical effort B. I sometimes (1-2 times last week) did physical things in my free time (eg. Played sports, went running, swimming, bike riding etc) C. I often (3-4 times last week) did physical things in my free time D. I quite often (5-6 times last week) did physical things in my free time E. I very often (7 or more times last week) did physical things in my free time 9. Mark how often you did physical activity (like playing sports, games, dancing or any other physical activity) for each day last week. (Tick one box per row) None A little bit Medium Often Very often Monday Tuesday Wednesday Thursday Friday Saturday 9. Mark how often you did physical activity (like playing sports, games, dancing or any other physical activity) for each day last week. (Tick one box per row) None A little bit Medium Often Very often Monday Tuesday Wednesday Thursday Friday Saturday 9. Mark how often you did physical activity (like playing sports, games, dancing or any other physical activity) for each day last week. (Tick one box per row) Physical activity in paediatric CMT Sunday 10. Were you sick last week, or did anything prevent you from doing your normal physical activities? (Tick on only) Yes If yes, what prevented you? • We would like to know about your level of physical activity from the last 7 days (the last week). ___________________________________ No Adapted from Kowalski KC, Crocker PRE, Donen RM. The Physical Activity Questionnaire for Older Children (PAQ-C) and Adolescents (PAQ-A) Manual. Saskatoon, Canada: University of Saskatchewan; 2004. Physical activity in paediatric CMT Sunday 10. Were you sick last week, or did anything prevent you from doing your normal physical activities? (Tick on only) Yes If yes, what prevented you? ___________________________________ No Adapted from Kowalski KC, Crocker PRE, Donen RM. The Physical Activity Questionnaire for Older Children (PAQ-C) and Adolescents (PAQ-A) Manual. Saskatoon, Canada: University of Saskatchewan; 2004. Physical activity in paediatric CMT 10. Were you sick last week, or did anything prevent you from doing your normal physical activities? (Tick on only) Adapted from Kowalski KC, Crocker PRE, Donen RM. The Physical Activity Questionnaire for Older Children (PAQ-C) and Adolescents (PAQ-A) Manual. Saskatoon, Canada: University of Saskatchewan; 2004.
https://openalex.org/W2074442603	https://pdf.blucher.com.br/medicalproceedings/flebologia/SIF_51.pdf	Portuguese	null	Ultrassonografia Trans-operatória nas Ablações de Eixos Safénicos	Blucher Medical Proceedings	2,014	cc-by	252	Blucher Medical Proceedings Fevereiro de 2014, Número 1, Volume 1 Blucher Medical Proceedings Fevereiro de 2014, Número 1, Volume 1 V SIMPÓSIO INTERNACIONAL DE FLEBOLOGIA V INTERNATIONAL PHLEBOLOGY SYMPOSIUM Ferreira, J. 2013. Ultrassonografia Trans- operatória nas Ablações de Eixos Safénicos, p.78. In: Bastos, Francisco Reis. Anais do V Simpósio Internacional de Flebologia [Blucher Medical Proceedings n.1 v.1]. São Paulo: Blucher, 2014 http://dx.doi.org/10.5151/medpro-flebo-SIF_51 Ultrassonografia Trans-operatória nas Ablações de Eixos Safénicos Ferreira J.1 1 Porto Alegre – Brasil. E-mail: drjulioferreira@ibf.med.br Ferreira J.1 1 Porto Alegre – Brasil. E-mail: drjulioferreira@ibf.med.br Ferreira J.1 1 Porto Alegre – Brasil. E-mail: drjulioferreira@ibf.med.br O Ultrassom é uma ferramenta fundamental para o diagnóstico e tratamento das patologias vasculares. Sua disponibilidade no Bloco Cirúrgico permite ao Cirurgião Vascular realizar procedimentos ecoguiados e mínimamente invasivos com abso- luta segurança e precisão. (Bloqueios nervosos, Anestesia tumescente para safenectomia cirúrgica ou ablações térmicas, acompanhamento dos processos de ablação com Radiofrequência ou Laser, etc.). No campo das ablações endovasculares com laser, fórmulas matemá- ticas foram criadas para otimizar seus parámetros de utilização visando obter o melhor índice de sucesso nesta técnica, mas num pequeno núme- ro de pacientes foi possível observar que a ablação não foi completa. Com o auxilio do Ultrassom trans-operatório, e seguindo critérios ultrasonográficos e não fórmulas matemáticas podem-se ter certeza de que o processo de ablação foi concluído com sucesso. O objetivo desta apresentação será mostrar quais são os critérios ultrassonográficos de certeza que permitem afirmar de que o processo de ablação está completo. Palavras-chave: ultra-som, laser, radiofrequência, varizes. 78sIIPs 78sIIPs
https://openalex.org/W4287828205	https://zenodo.org/record/3713275/files/Soosiana_vol34_PGB.pdf	English	null	Chilostoma (Cingulifera) cingulatum (S. Studer, 1820) (Gastropoda: Pulmonata: Helicidae) new to the fauna of Hungary	Zenodo (CERN European Organization for Nuclear Research)	2,020	cc-by	2,078	2. Dísz tér 8., Budapest, H-1014, Hungary 3. Institute of Enzymology, Research Center of Natural Sciences, Budapest, H-1117, Hungary, Magyar tudósok zsákutcája 2. 4. Department of Zoology, Hungarian Natural History Museum, H-1088 Baross u. 13, Budapest, Hungary * Corresponding author, e-mail: pallgergely2@gmail.com Submitted on 13 November 2019, accepted on 8 December 2019. Abstract Chilostoma cingulatum (S. Studer, 1820) is reported for the ﬁ rst time in Hungary. A seemingly viable population, consisting of several living adult and juvenile specimens has been discovered in Budapest, on the ruins of the Church of Mary Magdalene of the Castle of Buda. The collected specimens could be identiﬁ ed only to the species level. However, they are most similar to Chilostoma cingulatum baldense (Rossmässler, 1839). It is unlikely that this introduced population will become an invasive pest due to its narrow ecological niche, limestone-rich rocks and walls. Chilostoma cingulatum (S. Studer, 1820) is reported for the ﬁ rst time in Hungary. A seemingly viable population, consisting of several living adult and juvenile specimens has been discovered in Budapest, on the ruins of the Church of Mary Magdalene of the Castle of Buda. The collected specimens could be identiﬁ ed only to the species level. However, they are most similar to Chilostoma cingulatum baldense (Rossmässler, 1839). It is unlikely that this introduced population will become an invasive pest due to its narrow ecological niche, limestone-rich rocks and walls. Key words: introduced species, viable population, rock-dwelling snail Key words: introduced species, viable population, rock-dwelling snail Soosiana 34: 12-17, 2020 Published online at www.mamat.hu 15 March 2020 Soosiana 34: 12-17, 2020 Published online at www.mamat.hu 15 March 2020 12 doi: 10.5281/zenodo.3701569 Kivonat A Chilostoma cingulatum (S. Studer, 1820) (alpesi sávoscsiga) első adatát közüljük Magyarországról. Egy életképesnek látszó populációt fedeztünk fel a Mária Magdolna-templom (Budai Vár egyik temploma) romjain, melyet több kifejlett és ﬁ atal példány alkotott. A példányokat csak fajszinten azonosítottuk, azonban megjegyezzük, hogy a Chilostoma cingulatum baldense (Rossmässler, 1839) alfajhoz hasonlítanak leginkább. A faj szűk ökológiai tűrőképessége miatt (csak mészben gazdag rakott és sziklafalakon képes életben maradni) valószínűtlen, hogy ez a faj kártékony invazívvá váljon. Kulcsszavak: behurcolt faj, életképes populáció, sziklalakó csiga 1. Plant Protection Institute, Centre for Agricultural Research, Herman Ottó Street 15, Budapest, H-1022, Hungary 1. Plant Protection Institute, Centre for Agricultural Research, Herman Ottó Street 15, Budapest, H-1022, Hungary 2. Dísz tér 8., Budapest, H-1014, Hungary Introduction The second author collected some snails on the ruins of the Church of Mary Magdalene of the Castle of Buda, Budapest, District I, Hungary. Through a review of the literature and comparisons of the shells with the specimens deposited in the Hungarian Natural History Museum (HNHM) the snails were identiﬁ ed as Chilostoma cingulatum (S. Studer, 1820), a species normally inhabiting the Northern Alps (Liechtenstein to Salzburg), the Southern Alps (Ticino, Switzerland), and the Apennines Monte del Matese, Italy) (Pfeiffer 1951, Welter-Schultes 2012). This species has been introduced to a number of German urban areas (Schlesch 1962, Fauer 1998, Kobialka 2000, Schmid 2000, Rosenbauer 2011, Hirschfelder 2017, Kittel 2017, Eta & Hausdorf 2019) and Austria near Vienna (Fischer et al. 2010). This is its ﬁ rst record from Hungary, extending its area considerably eastwards. Soosiana 34: 12-17, 2020 13 Collection site (H. Beck, 1837) (anatomically veriﬁ ed) were also collected at the site. Budapest, Várnegyed, ruins of Church of Mary Magdalene, southern side, ca. 47°30.222’N, 19°1.734’E (Fig. 1.). Specimens were found at two sites where grass was not cut after rain on 26 August 2019. At one site 26 adult and 6 juvenile specimens were collected, and 15 adults and 6 juveniles were found at the other. Living specimens of Oxychilus draparnaudi Three specimens (shells and ethanol- preserved bodies) are deposited in the HNHM (HNHM 104406: ﬁ gured specimen; HNHM 104407: two additional specimens). Figure 1. A–B: Living Chilostoma cingulatum (S. Studer, 1820); C–D: habitat of Chilostoma cingulatum at the Church of Mary Magdalene, Várnegyed, Budapest. Photos: Natália Tőkési. Figure 1. A–B: Living Chilostoma cingulatum (S. Studer, 1820); C–D: habitat of Chilostoma cingulatum at the Church of Mary Magdalene, Várnegyed, Budapest. Photos: Natália Tőkési. Páll-Gergely et al.: Chilostoma cingulatum new to the fauna of Hungary 14 Family Helicidae Raﬁ nesque, 1815 Subfamily Ariantinae Mörch, 1864 Genus Chilostoma Fitzinger, 1833 Chilostoma cingulatum (S. Studer, 1820) belongs to the monotypic subgenus Cingulifera Held, 1838 (Groenenberg et al. 2016). This species had the highest number of described subspecies among European land snails (Bank 2007) with 29 accepted subspecies. Two of them were elevated to species level (Groenenberg et al. 2016), thus, 27 subspecies remained (Molluscabase 2019). This extreme splitting is probably due to the large, fragmented distribution (one of the largest in this genus), and that taxonomists tend to describe more subspecies for taxa having larger and more character-rich shells (Páll-Gergely et al. 2019). The populations found in Budapest is most similar to Chilostoma cingulatum baldense (Rossmässler, 1839) (B. Hausdorf, pers. comm., 2019 December). However, since intraspeciﬁ c systematics of this species is not resolved yet, we identify the newly discovered population to species level only. Future research, especially molecular phylogeny, could shed light on the origin of the Hungarian population. Chilostoma (Cingulifera) cingulatum (S. Studer, 1820) Chilostoma (Cingulifera) cingulatum (S. Studer, 1820) Description of the shell (Fig. 2): Depressed, with only slightly elevated spire, dorsal side domed, body whorl rounded, consisting of 4.25–4.75 whorls; base colour light brown to whitish with a slender, reddish brown peripheral band; shell surface glossy, almost smooth, with ﬁ ne, inconspicuous, irregular growth lines; aperture semilunar, strongly oblique to shell axis; peristome strongly expanded, not reﬂ ected, except in direction of the umbilicus; umbilicus open, narrow, its edge is covered by reﬂ ected peristome. The populations found in Budapest is most similar to Chilostoma cingulatum baldense (Rossmässler, 1839) (B. Hausdorf, pers. comm., 2019 December). However, since intraspeciﬁ c systematics of this species is not resolved yet, we identify the newly discovered population to species level only. Future research, especially molecular phylogeny, could shed light on the origin of the Hungarian population. band; shell surface glossy, almost smooth, with ﬁ ne, inconspicuous, irregular growth lines; aperture semilunar, strongly oblique to shell axis; peristome strongly expanded, not reﬂ ected, except in direction of the umbilicus; umbilicus open, narrow, its edge is covered by reﬂ ected peristome. Figure 2. Chilostoma cingulatum (S. Studer, 1820) found on the ruins of the Church of Mary Magdalene, Várnegyed, Budapest (HNHM 104406). Scale bar represents 20 mm. Photos: B. Páll-Gergely. Figure 2. Chilostoma cingulatum (S. Studer, 1820) found on the ruins of the Church of Mary Magdalene Várnegyed, Budapest (HNHM 104406). Scale bar represents 20 mm. Photos: B. Páll-Gergely. Figure 2. Chilostoma cingulatum (S. Studer, 1820) found on the ruins of the Church of Mary Magdalene, Várnegyed, Budapest (HNHM 104406). Scale bar represents 20 mm. Photos: B. Páll-Gergely. Soosiana 34: 12-17, 2020 15 Description of the genitalia (Fig. Discussion The presence of living adult and juvenile specimens indicates that a viable population of Chilostoma cingulatum is established at the ruins of the Church of Mary Magdalene, Várnegyed, Budapest. Since this species is an obligate limestone-dweller in its native range (Welter- Schultes 2012), and wherever it has been introduced (Eta & Hausdorf 2019), we speculate that the species will not disperse beyond man- made limestone walls. Consequently, it seems unlikely that this introduced species will become a highly invasive pest. Chilostoma (Cingulifera) cingulatum (S. Studer, 1820) 3): Atrium short, internally with an atrial stimulator; penis short, penial verge conical with an elongated tip, opening laterally at the base of tip; epiphallus cylindrical, relatively slender, approximately twice as long as penis; retractor muscle inserts near middle of epiphallus, slightly in distal direction; ﬂ agellum slender, extremely long (at least twice as long as penis and epiphallus combined), vermiform; vas deferens as thick as ﬂ agellum, moderately long; vagina slightly thicker and longer than penis; dart sac short, oval; dart with a slender stalk and a wider blade; glandulae mucosae two, simple, relatively thick, at least 5 times longer than dart sac; pedunculus branches to bursa copulatrix and diverticulum; stalk of bursa copulatrix at least twice as long as pedunculus; bursa small, rounded; diverticulum approximately 3.5 times as long as pedunculus; end of diverticulum not tapering; diverticulum attached to spermoviduct with numerous ﬁ bres; albumen gland long (slightly longer than spermoviduct), thick, C-shaped; talon clearly visible, relatively weakly developed. Differential diagnosis: Chilostoma cingulatum is not similar to any other land snail species reported from Budapest. The most similar species from Hungary is Faustina faustina (Rossmässler, 1835), which is known from the North Hungarian Mountains (Subai & Neubert 2016). That species has a more elevated spire, a wider umbilicus, and its yellow colour morph has a darker colouration.oreover, the spiral band, which is not present in all F. faustina specimens, is thicker than that of C. cingulatum. Acknowledgements We are grateful to László Badacsonyi and Gábor Majoros for their ﬁ eld observations at the collection site, and to Alexander Reischütz and Bernhard Hausdorf for their comments on the manuscript. Páll-Gergely et al.: Chilostoma cingulatum new to the fauna of Hungary 16 3. Reproductive anatomy of Chilostoma cingulatum (S. Studer, 1820) (HNHM 104406). Abbr bumen gland; bc: bursa of the bursa copulatrix; cf: connecting ﬁ laments; d: diverticulum; ds: iphallus; f: ﬂ agellum; gm: glandulae mucosae; go: genital opening; hd: hermaphrodi nis; pe: pedunculus; rm: retractor muscle; so: spermoviduct, t: talon; v: vagina; vd: vas ar represents 5 mm. Figure 3. Reproductive anatomy of Chilostoma cingulatum (S. Studer, 1820) (HNHM 104406). Abbreviations: ag: albumen gland; bc: bursa of the bursa copulatrix; cf: connecting ﬁ laments; d: diverticulum; ds: dart sac; e: epiphallus; f: ﬂ agellum; gm: glandulae mucosae; go: genital opening; hd: hermaphroditic duct; p: penis; pe: pedunculus; rm: retractor muscle; so: spermoviduct, t: talon; v: vagina; vd: vas deferens. Scale bar represents 5 mm. Figure 3. Reproductive anatomy of Chilostoma cingulatum (S. Studer, 1820) (HNHM 104406). Abbreviations: ag: albumen gland; bc: bursa of the bursa copulatrix; cf: connecting ﬁ laments; d: diverticulum; ds: dart sac; e: epiphallus; f: ﬂ agellum; gm: glandulae mucosae; go: genital opening; hd: hermaphroditic duct; p: penis; pe: pedunculus; rm: retractor muscle; so: spermoviduct, t: talon; v: vagina; vd: vas deferens. Scale bar represents 5 mm. Soosiana 34: 12-17, 2020 17 References BANK, R.A. (2007). Mollusca: Gastropoda. Fauna Europaea version 2.4. 2013 September 27. Retrieved from https://www.faunaeur.org MOLLUSCABASE (2019). MolluscaBase. Chilostoma cingulatum (Studer, 1820). Accessed at: http://www.molluscabase.org/aphia. php?p=taxdetails&id=871135 on 2019-12-23 ETA, K. & HAUSDORF, B. (2019): Chilostoma cingulatum (Studer 1820) und Cornu aspersum (O. F. Müller 1774) in Hamburg (Gastropoda: Helicidae). — Mitteilungen der Deutschen Malakozoologischen Gesellschaft, 100: 13–16. PÁLL-GERGELY, B., ASAMI, T. & SÓLYMOS, P. (2019): Subspecies description rates are higher in morphologically complex land snails. — Zoologica Scripta, 48: 185–193. PFEIFFER, K.L. (1951): Chilostoma (Cingulifera) cingulata (Studer). Versuch einer monographischen Darstellung des Rassenkreises. — Archiv für Molluskenkunde, 80(4/6): 89–214, Tafel 5–10. FAUER, W. (1998): Zum Vorkommen der Großen Felsenschnecke Chilostoma cingulatum (S. Studer 1820) im mittleren und nördlichen Bayern (Gastropoda: Helicidae). — Heldia, 2(5/6): 137–140. ROSENBAUER, A. (2011): Vorkommen südeuropäischer Schneckenarten in Steinmetzbetrieben. — Mitteilungen der Deutschen Malakozoologischen Gesellschaft, 85: 27–34. FISCHER, W., REISCHÜTZ, A. & REISCHÜTZ, P.L. (2010): Beiträge zur Kenntnis der österreichischen Molluskenfauna XIX. Acknowledgements Es kam nicht nur der Marmor nach Wien – Biodiversität auf dem Gelände eines ehemaligen Steinmetzbetriebes. — Nachrichtenblatt der Ersten Vorarlberger Malakologischen Gesellschaft, 17: 9–12. SCHMID, G. (2000): Die Große Felsenschnecke Chilostoma cingulatum (Studer) an Buntsandsteinmauern im Nordschwarzwald. — Carolinea, 58: 149–154, Taf. 1. GROENENBERG, D.S.J., SUBAI, P. & GITTENBERGER, E. (2016): Systematics of Ariantinae (Gastropoda, Pulmonata, Helicidae), a new approach to an old problem. — Contributions to Zoology, 85 (1): 37–65. SCHLESCH, H. (1962): Bemerkungen und Berichtigungen zum neuerschienenen Ergänzungsband Mollusken in die Tierwelt Mitteleuropas, Band 2, Lfg. 1 (1962). — Mitteilungen der Deutschen Malakozoologischen Gesellschaft, 1(2): 24–26. HIRSCHFELDER, H.-J. (2017): Zwei weitere außeralpine Vorkommen der Großen Felsenschnecke (Chilostoma cingulatum) in Bayern. — Mitteilungen der Deutschen Malakozoologischen Gesellschaft, 97: 79–81. SUBAI, P. & NEUBERT, E. (2016): Revision of the Ariantinae. 4. The genus Faustina Kobelt 1904 (Gastropoda: Pulmonata: Helicidae). — Archiv für Molluskenkunde, 145(1): 85–110. KITTEL, K. (2017): Die Weichtierfauna mittelalterlicher KITTEL, K. (2017): Die Weichtierfauna mittelalterlicher und frühneuzeitlicher Ruinen Unterfrankens. — Mitteilungen des Naturwissenschaftlichen Museums der Stadt Aschaffenburg, 28: 1—360. WELTER-SCHULTES, F.W. (2012): European non- marine molluscs, a guide for species identiﬁ cation. — pp. A1–A3, 1–679, Q1–Q78, Göttingen. (Planet Poster Editions). KOBIALKA, H. (2000): Zum Vorkommen der Großen Felsenschnecke Chilostoma cingulatum (S. Studer 1820) in Baden-Württemberg (Gastropoda: Helicidae). — Mitteilungen der Deutschen Malakozoologischen Gesellschaft, 65: 45–49.
https://openalex.org/W1970378131	https://europepmc.org/articles/pmc3271104?pdf=render	English	null	CCL2-Expressing Astrocytes Mediate the Extravasation of T Lymphocytes in the Brain. Evidence from Patients with Glioma and Experimental Models In Vivo	PloS one	2,012	cc-by	8,311	Abstract CCL2 is a chemokine involved in brain inflammation, but the way in which it contributes to the entrance of lymphocytes in the parenchyma is unclear. Imaging of the cell type responsible for this task and details on how the process takes place in vivo remain elusive. Herein, we analyze the cell type that overexpresses CCL2 in multiple scenarios of T-cell infiltration in the brain and in three different species. We observe that CCL2+ astrocytes play a part in the infiltration of T-cells in the brain and our analysis shows that the contact of T-cells with perivascular astrocytes occurs, suggesting that may be an important event for lymphocyte extravasation. Citation: Carrillo-de Sauvage MA, Go´mez A, Ros CM, Ros-Bernal F, Martı´n ED, et al. (2012) CCL2-Expressing Astrocytes Mediate the Extravasation of T Lymphocytes in the Brain. Evidence from Patients with Glioma and Experimental Models In Vivo. PLoS ONE 7(2): e30762. doi:10.1371/journal.pone.0030762 Editor: Pablo Villoslada, Institute Biomedical Research August Pi Sunyer (IDIBAPS) - Hospital Clinic of Barcelona, Spain Editor: Pablo Villoslada, Institute Biomedical Research August Pi Sunyer (IDIBAPS) - Hospital Clinic of Barcelona, Spain Received May 12, 2011; Accepted December 27, 2011; Published February 2, 2012 Copyright: 2012 Carrillo-de Sauvage 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 grants from: the Spanish Ministry of Science and Innovation (FIS PI10 02827, SAF 2004 07656 C02-02, SAF 2010 21274, RYC- 2010-06729), Fundacio´n Se´neca (FS/15329/PI/10) and CIBERNED (Centro de Investigacio´n Biome´dica en Red sobre Enfermedades Neurodegenerativas). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: mtherrer@um.es . These authors contributed equally to this work. . These authors contributed equally to this work. effect on T lymphocytes. Significantly, CCL2 induces the formation of the uropod in T-cells [1], a cellular formation crucial for the cell’s entrance into the parenchyma. However, identifica- tion of the brain cells in charge of this phase of extravasation in the CNS and data on how the process takes place remain elusive. In the present work we analyze the cellular expression of CCL2 in three independent scenarios of T-cell infiltration in the brain in vivo. Abstract We observe that the perivascular astrocytes highly express CCL2, independently of the species and the inflammatory situation, facilitating the entrance of lymphocytes into the brain. CCL2-expressing astrocytes are specifically localized in the T-cell- infiltration areas independently of the inflammatory scenario. Importantly, blocking CCL2 attenuates the penetration of T-cell infiltration in the brain, which suggests that CCL2 produced by reactive astrocytes at the site of inflammation is responsible for T- cell extravasation. On the other hand, the physical contact between CCL2+ astrocytes and CD3+ T-cells takes place at perivascular areas of the brain and we hypothesized that this phenomenon may be important for lymphocyte internalization. CCL2-Expressing Astrocytes Mediate the Extravasation of T Lymphocytes in the Brain. Evidence from Patients with Glioma and Experimental Models In Vivo Maria Angeles Carrillo-de Sauvage1,2., Aurora Go´ mez1,2., Carmen Marı´a Ros1,2, Francisco Ros-Bernal1,2, Eduardo D. Martı´n3, Ana Perez-Valle´s5, Jose´ M. Gallego-Sanchez4, Emiliano Ferna´ndez-Villalba1,2, Carlos Barcia Sr.4, Carlos Barcia Jr.1,2, Maria-Trinidad Herrero1,2* 1 Clinical and Experimental Neuroscience, University of Murcia, Murcia, Spain, 2 Centro de Investigacio´n Biome´dica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), School of Medicine, University of Murcia, Murcia, Spain, 3 Instituto de Investigacio´n en Discapacidades Neurolo´gicas (IDINE), University of Castilla-La Mancha, Albacete, Spain, 4 Department of Neurosurgery, Hospital General Universitario de Valencia, Valencia, Spain, 5 Department of Pathology, Hospital General Universitario de Valencia, Valencia, Spain Introduction Lymphocyte infiltration is an important phenomenon in the inflammatory response and is an event involved in many degenerative scenarios of the CNS. Many brain tissue diseases, such as brain tumors and viral or bacterial infections, among many others, show infiltrated T-cell subsets. The effect of such T-cell infiltration may vary in different CNS diseases, but controlling the entrance of blood cells may have important clinical and therapeutic implications. The phenomenon of extravasation is regulated by a cascade of molecular events that involves the adhesion of lymphocytes to the endothelium following their migration into tissue [1,2,3]. Selectin-mediated interactions cause lymphocytes to roll over the endothelial cells, at the same time coming into contact different factors. The adhesion molecules involved in the recruitment of lymphocytes into the tissue (such as ICAM-1, VCAM or LFA-1) are well defined but less is known about the factors that trigger the adhesion and direct the extravasation. It has been proposed that chemokines or chemoat- tractive cytokines trigger the adhesion and migration of lympho- cytes either as soluble chemoattractants or as immobilized molecules bound to proteoglycans of the endothelial surface [4]. CCL2 (formerly MCP-1) is one of the most important chemokines involved in the recruitment of blood cells, especially macrophages [5], into the tissues but fewer information is reported about its Mice Twenty-seven adult male C57BL6 mice were used to test the role of CCL2 in the infiltration of lymphocytes in the brain. Fourteen mice were used in a first experiment to analyze the correlation between the infiltration of T-cells and the expression of CCL2 cells after LPS injection. On day 0, mice were anesthetized using ketamine (50 mg/kg) and medetomidine (1 mg/kg) and either 2 mg/ml of LPS (n = 7) or saline (n = 7) in 1 ml of volume was injected into the right striatum as previously described [8]. Four days were allowed to pass waiting for the immune response to be stimulated. Then, animals were sacrificed on day 7, at the peak of the potential T-cell response as has been previously described [9]. On the other hand, in order to know the effect of CCL2 in the lymphocyte infiltration, another experiment was performed injecting goat anti-mouse CCL2 neutralizing antibody (R&D Systems) directly into the brain parenchyma. Mice were anesthetized and placed in the stereotactic frame and an injecting guide cannula (Plastics One, C315G-SPC, Roanoke, VA, USA) was placed in the right striatum in the same coordinates indicated above. The guide cannula was then protected with a dummy cannula (Plastics ONE, C315CD-SPC) following the instructions of the manufacturers. After surgery, animals were left in their cage for 15 days in order to eliminate/diminish the inflammation caused by the intra-parenchymal placement of the cannula. After this time, animals received an injection of LPS (2 mg/ml) using an internal cannula (Plastics ONE, C3151-SPC), through the previously implanted guided cannula, and four days later they received, through the same cannula and with the help of a microinjector, either an intraparenchymal injection of goat anti- mouse CCL2 neutralizing antibodies (0.1 mg/ml, n = 3 or 1 mg/ml, n = 5) or the purified isotype immunoglobulin (Goat IgG, 0.1 mg/ Quantification and sterological analysis The number of labeled cells was quantified as previously described [10]. CCL2+ cells and CD3+ T-cells were quantified in all species studied. In mice, the number of CD4+ and CD8+ T-cells was also counted. Correlations between the number of CCL2+ cells and the number of CD3+ cells were calculated from the data obtained from the quantifications of DAB+ immunostained sections. To obtain the data for the correlations, two independents stereological quantifica- tions of CD3 and CCL2 were performed in serial adjacent sections in all specimens. Results were expressed as the mean 6 SEM. Confocal imaging Brain sections from human biopsies, monkey and mouse brain were examined by confocal microscopy as described previously [7]. Images are shown as the transparency of the stack of images or showing a single 0.5 mm optical section. Patients and Samples All the experiments were conducted in accordance with the Helsinki Declaration, the Guide for the Care and Use of Laboratory Animals (NIH Guide, revised 1996), the Guidelines of the Europeanonvention for the protection of Vertebrate Animals used for Experimental and other scientific purposes of the Council of Europe (no 123, June 15th, 2006) and the European Communities Council Directive 2010/63/ECC. Immunocytochemical procedures All primate samples were collected for previously published studies [7]. In the present work, we analyzed the brain tissue sections from seven adult (either male or female) cynomolgus monkeys (Macaca fascicularis) (5 kg body weight) that were processed in preceding studies [7]. Briefly, the animals were injected bilaterally with 16107 infective units of Ad-mCMV-bgal adenovirus (Kindly provided by Dr. P.R. Lowenstein and Dr. M.G. Castro) in six brain coordinates, including cortex and white matter and the tissue was processed as described previously [7]. In all studies involving non-human primates, all necessary steps were taken to ameliorate any suffering or pain. The referenced study was carried out according to the approved protocols of the University of Murcia (Project ID: FEDER FD1/97-1931) and all the experiments were conducted in accordance with the Guidelines of the European Convention for the protection of Vertebrate Animals used for Experimental and other scientific purposes of the Council of Europe (nu 123, June 15th 2006). Brain tumor sections (60 mm) were selected according to the anatomopathological criteria (Figure S1), cut serially through the entire sample, and immunofluorescence or diaminobenzidine (DAB) detection was performed as described previously [6]. Coronal monkey brain sections (50 mm) were cut serially through the entire brain, and DAB detection or immunofluorescence was performed as described previously [7]. In addition, hematoxilin staining was performed in some of the sections to confirm the localization of tumorigenic areas and blood vessels (BV). For human or monkey tissue the following primary antibodies were used: anti-human CCL2 (MCP-1) (1:40, mouse IgG2b; R&D), anti-human CD3 (1:100, Rabbit or 1:50, Mouse, IgG1; Dako), anti-human CD20 (1:500, mouse, IgG2a; Dako), anti-GFAP (1:500, Rabbit; Chemicon), anti-LFA-1 (1:50, Rabbit; Abcam), anti-human ICAM-1 (1:100, Mouse, IgG1; Sigma-Aldrich), anti- human VCAM-1 (1:50, mouse, IgG1k; Dako), anti-CCR2 (1:200, goat; Abcam) and Collagen-IV (1:500, Rabbit; Abcam). Mouse brains were sectioned in 30 mm serial sections using a vibratome (Leica), and were stained for immunofluorescence or DAB detection using previously published protocols [8]. The following antibodies were used: anti-mouse CCL2 (1:200, Rabbit, Serotec), anti-mouse CD4 (1:750, Rat; Serotec), anti-mouse CD8 (1:750, Rat; Serotec), anti-mouse CD3 (1:750, Hamster, Serotec) and anti- GFAP (1:500; mouse, Chemicon, Millipore). Patients and Samples Fourteen patients from the General Hospital of Valencia (Spain) were diagnosed with glioma of different degrees and were cited for PLoS ONE \| www.plosone.org February 2012 \| Volume 7 \| Issue 2 \| e30762 1 Astrocytes Mediate T-Cell Infiltration ml) on day 4th and 6th. Seven days after the LPS injection animals were anesthetized and sacrificed and processed as described below. ml) on day 4th and 6th. Seven days after the LPS injection animals were anesthetized and sacrificed and processed as described below. Mice were anesthetized with an overdose of ketamine/xylacine for perfusion-fixation (with PBS followed by 4% PFA). Brain tissue was then removed and post-fixed in PFA for 48 hours. The study was carried out according to the approved protocols of the University of Murcia (Project ID: FIS/PI10/02827). All the experiments were conducted in accordance with the Helsinki Declaration, the Guide for the Care and Use of Laboratory Animals (NIH Guide, revised 1996), the Guidelines of the Europeanonvention for the protection of Vertebrate Animals used for Experimental and other scientific purposes of the Council of Europe (no 123, June 15th, 2006) and the European Communities Council Directive 2010/63/ECC. intracranial surgery. The fourteen cases showed the typical features of astrocytoma, with the classic clinical evolution, neuroimaging, spectroscopy, and anatomo-pathological examina- tion (Figure S1). The study was carried out according to the approved protocols of all the institutions involved (General Hospital of Valencia and University of Murcia). The approval from the IRB of the University of Murcia was received regarding the project (RYC-2010-06729) and a written informed consent was obtained from all participants involved in the study (General Hospital Permits # 07B0002707-09B0010238; University of Murcia Project ID: RYC-2010-06729). The brain tumors presented characteristic glioma morphology. Tumors were excised and processed with a previously published protocol [6]. Mice were anesthetized with an overdose of ketamine/xylacine for perfusion-fixation (with PBS followed by 4% PFA). Brain tissue was then removed and post-fixed in PFA for 48 hours. The study was carried out according to the approved protocols of the University of Murcia (Project ID: FIS/PI10/02827). Results single cells showed star shape structures (Figure 1A, images 20– 24) similar to astrocyte-specific GFAP staining (Figure 1B). BVs, inside and close to tumorigenic areas, strongly expressed CCL2, but did not do so outside the tumorigenic areas (Figure 1C). Importantly, confocal co-localization analysis revealed that astro- cytes were the CCL2-expressing cells in the tumor area (Figure 2A). single cells showed star shape structures (Figure 1A, images 20– 24) similar to astrocyte-specific GFAP staining (Figure 1B). BVs, inside and close to tumorigenic areas, strongly expressed CCL2, but did not do so outside the tumorigenic areas (Figure 1C). Importantly, confocal co-localization analysis revealed that astro- cytes were the CCL2-expressing cells in the tumor area (Figure 2A). These CCL2+ areas were seen to present the specific infiltration of CD3+ T-cells in brain tumors (Figure 2B). However, in order to understand the role of CCL2 in the specific extravasation of CD3+ lymphocytes, the correlation between the number of tumor infiltrating CD3+ T-cells and the number of expressing CCL2 cells in the glioma samples was calculated. Importantly, we divided the All biopsies of patients with glioma presented high expression of CCL2 in tumorigenic areas (Figure 1A). Importantly, all the cases studied showed CCL2 immunoreactive cells, while the shape of the areas of immunoreactivity differed according to the structure of the tumor (Figure 1A, images 1–24). Areas far from the tumorigenic center, putatively healthy tissue, did not show CCL2 immunore- activity (Figure 1A, image 3) and a gradient of immunoreactivity from non tumorigenic to tumorigenic areas could be clearly seen in most cases (Figure 1A, image 10). CCL2 was especially high around BVs’ lumen (Figure 1A, images 16–19 and 169–199) and These CCL2+ areas were seen to present the specific infiltration of CD3+ T-cells in brain tumors (Figure 2B). However, in order to understand the role of CCL2 in the specific extravasation of CD3+ lymphocytes, the correlation between the number of tumor infiltrating CD3+ T-cells and the number of expressing CCL2 cells in the glioma samples was calculated. Importantly, we divided the Figure 1. Human glioma shows CCL2+ cells. (A) The 14 cases of glioma analyzed show CCL2 expression in the tumorigenic areas. Samples of liomas were immunostained to detect the expression of CCL2. All 14 cases analyzed showed expression of CCL2 in the neoplasic areas. CCL2- xpressing cells are localized in the brain parenchyma itself and their number and intensity increase towards the necrotic areas. Statistical analysis Data are expressed as mean 6 SEM. Statistical analysis was performed using student’s t-test or one-way ANOVA test following a posthoc analysis. The null hypothesis was rejected for an a risk equal to 5%. PLoS ONE \| www.plosone.org February 2012 \| Volume 7 \| Issue 2 \| e30762 February 2012 \| Volume 7 \| Issue 2 \| e30762 2 Astrocytes Mediate T-Cell Infiltration Results CCL2-expressing cells an also be seen around BVs (16–19). In addition, counterstaining with hematoxilin is also shown at BV levels (169–199) to corroborate the presence of ndothelial nuclei. Insert show a detail of the endothelial nuclei. Scale bar; 1–21: 400 mm, 22–24: 60 mm. (B) Tumor cells present high mmunoreactivity for GFAP in tumor areas, demonstrating the typical astrocytic cell type. CCL2+ cells show a characteristic astrocytic morphology. cale bar: 30 mm. (C) BVs in, or close to the putative tumorigenic areas show high immunoreactivity for CCL2 in contrast to normal tissue. Additionally, counterstaining with hematoxilin (HHS) is also shown at the same levels. The insert shows a detail of the endothelial nuclei. Scale bar: 00 mm. oi:10.1371/journal.pone.0030762.g001 Figure 1. Human glioma shows CCL2+ cells. (A) The 14 cases of glioma analyzed show CCL2 expression in the tumorigenic areas Samples of Figure 1. Human glioma shows CCL2+ cells. (A) The 14 cases of glioma analyzed show CCL2 expression in the tumorigenic areas. Samples of gliomas were immunostained to detect the expression of CCL2. All 14 cases analyzed showed expression of CCL2 in the neoplasic areas. CCL2- expressing cells are localized in the brain parenchyma itself and their number and intensity increase towards the necrotic areas. CCL2-expressing cells can also be seen around BVs (16–19). In addition, counterstaining with hematoxilin is also shown at BV levels (169–199) to corroborate the presence of endothelial nuclei. Insert show a detail of the endothelial nuclei. Scale bar; 1–21: 400 mm, 22–24: 60 mm. (B) Tumor cells present high immunoreactivity for GFAP in tumor areas, demonstrating the typical astrocytic cell type. CCL2+ cells show a characteristic astrocytic morphology. Scale bar: 30 mm. (C) BVs in, or close to the putative tumorigenic areas show high immunoreactivity for CCL2 in contrast to normal tissue. Additionally, counterstaining with hematoxilin (HHS) is also shown at the same levels. The insert shows a detail of the endothelial nuclei. Scale bar: 100 mm. doi:10 1371/journal pone 0030762 g001 m doi:10.1371/journal.pone.0030762.g001 PLoS ONE \| www.plosone.org February 2012 \| Volume 7 \| Issue 2 \| e30762 3 Astrocytes Mediate T-Cell Infiltration T-cells into two different populations: T-cells inside the BV lumen d h h d (Fi 2C) I i l suggest that the presence of CCL2+ cells may be important for h i f l h l h h h i ff f Figure 2. CCL2+ cells are GFAP+ astrocytes in human glioma. Results (A) Confocal images show co-localization of CCL2-expressing cells (red) with GFAP (green) in glioma. Insert shows a detail of a single CCL2-expressing cell (red) co-localizing with GFAP (green) and counterstained with DAPI (blue). Scale bar: 30 mm. (B) Areas of infiltration of CD3+ T-cells (green) coincide with the areas of CCL2-expressing cells (red) in glioma. Confocal images show a tumorigenic area, infiltrated with CD3+ T-cells (green) in an area with numerous CCL2-expressing cells (red). The immunostaining was combined with a counterstaining with DAPI to detect the nucleus (blue). Scale bar: 50 mm. (C) Examples of characteristic CD3 infiltration in samples of glioma. CD3+ T-cells can be observed grouped in BVs (BV, limited by broken red line in 1 and 3) but also infiltrated in the parenchyma (2, 4, blue insert magnified in 5). (D) CCL2 expression correlates with the infiltration of T-cells in the tumor areas. The quantification in serial sections of the number of T-cells, either infiltrated or located in the BV lumen, revealed that the level of infiltration of CD3+ T-cells in the parenchyma (green) is positively correlated with the level of CCL2 expression (BV; BV lumen). doi:10.1371/journal.pone.0030762.g002 Figure 2. CCL2+ cells are GFAP+ astrocytes in human glioma. (A) Confocal images show co-localization of CCL2-expressing cells (red) with GFAP (green) in glioma. Insert shows a detail of a single CCL2-expressing cell (red) co-localizing with GFAP (green) and counterstained with DAPI (blue). Scale bar: 30 mm. (B) Areas of infiltration of CD3+ T-cells (green) coincide with the areas of CCL2-expressing cells (red) in glioma. Confocal images show a tumorigenic area, infiltrated with CD3+ T-cells (green) in an area with numerous CCL2-expressing cells (red). The immunostaining was combined with a counterstaining with DAPI to detect the nucleus (blue). Scale bar: 50 mm. (C) Examples of characteristic CD3 infiltration in samples of glioma. CD3+ T-cells can be observed grouped in BVs (BV, limited by broken red line in 1 and 3) but also infiltrated in the parenchyma (2, 4, blue insert magnified in 5). (D) CCL2 expression correlates with the infiltration of T-cells in the tumor areas. The quantification in serial sections of the number of T-cells, either infiltrated or located in the BV lumen, revealed that the level of infiltration of CD3+ T-cells in the parenchyma (green) is positively correlated with the level of CCL2 expression (BV; BV lumen). doi:10.1371/journal.pone.0030762.g002 Figure 2. CCL2+ cells are GFAP+ astrocytes in human glioma. Results Importantly, the areas of CD3+ T-cell infiltration show CCL2 expressing cells (Figure 3E) and a positive correlation was found between the number of infiltrated CD3+ T-cells and the number of CCL2+ cells in the adenoviral injected areas of monkey brain. Figure 3. CCL2+ astrocytes are expressed in T-cell infiltration areas in monkey brain. (A) Immunofluorescence shows the specific CCL2 expression located at the area of adenoviral injection in the macaque brain. Drawing on the right shows the anatomical location of the stereotaxic injection in the brain cortex. (B) Confocal images of BVs in an intact area and an injected area of the monkey brain. Perivascular astrocytes marked with GFAP (green) show CCL2 expression (red) in the adenoviral-injected areas. DAPI (blue) was used as a nuclear counterstaining to show the peculiar disposition of endothelial nuclei. (C) CCL2 is expressed by astrocytes in inflamed monkey brain. The staining of CCL2 (red) colocalizes with the astrocyte marker GFAP (green) in monkey cortex in the adenovirally injected area. (D) Three dimensional transparency of an area of CD3+ T-cells (green) infiltration shows specific CCL2 expression (magenta) in the brain of adenoviral injected monkey. DAPI was used as a counterstaining. Details of the insert are shown in the images on the right. (E) The stereological quantification of serial adjacent sections demonstrated that the number of infiltrated CD3+ cells correlates with CCL2+ cells in adenoviral injected areas in monkey brain. doi:10.1371/journal.pone.0030762.g003 Figure 3. CCL2+ astrocytes are expressed in T-cell infiltration areas in monkey brain. (A) Immunofluorescence shows the specific CCL2 expression located at the area of adenoviral injection in the macaque brain. Drawing on the right shows the anatomical location of the stereotaxic injection in the brain cortex. (B) Confocal images of BVs in an intact area and an injected area of the monkey brain. Perivascular astrocytes marked with GFAP (green) show CCL2 expression (red) in the adenoviral-injected areas. DAPI (blue) was used as a nuclear counterstaining to show the peculiar disposition of endothelial nuclei. (C) CCL2 is expressed by astrocytes in inflamed monkey brain. The staining of CCL2 (red) colocalizes with the astrocyte marker GFAP (green) in monkey cortex in the adenovirally injected area. (D) Three dimensional transparency of an area of CD3+ T-cells (green) infiltration shows specific CCL2 expression (magenta) in the brain of adenoviral injected monkey. DAPI was used as a counterstaining. Details of the insert are shown in the images on the right. Results Importantly, the areas of CD3+ T-cell infiltration show CCL2 expressing cells (Figure 3E) and a positive correlation was found between the number of infiltrated CD3+ T-cells and the number of CCL2+ cells in the adenoviral injected areas of monkey brain. tissue from monkeys that had been infected stereotactically in the cerebral cortex with artificially engineered adenoviral vectors. Previous published analyses have shown that adenoviral injections in the CNS elicit a specific infiltration of lymphocytes restricted to the injection areas [7]. We analyzed the presence of lymphocytes in the areas of injection, which were seen to express ICAM-1, VCAM (Figures S2 and S3) in contrast with intact areas (Figure S4). In addition, infiltrating lymphocytes express LFA-1 in the extravasation areas (Figure S5). Moreover, these areas of viral injection showed a particular expression of CCL2 limited to the injection area (Figure 3A). Importantly, confocal analysis of the infiltrated areas showed highly immunoreactive CCL2- expressing cells around BVs that were not present in intact (non- injected) areas (Figure 3B, C and Figure S6). In addition, detailed analysis of the virally injected areas demonstrated co- localization of CCL2+ and GFAP+ cells (Figure 3D). Importantly, the areas of CD3+ T-cell infiltration show CCL2 expressing cells (Figure 3E) and a positive correlation was found between the number of infiltrated CD3+ T-cells and the number of CCL2+ cells in the adenoviral injected areas of monkey brain. the injection area (Figure 3A). Importantly, confocal analysis of the infiltrated areas showed highly immunoreactive CCL2- expressing cells around BVs that were not present in intact (non- injected) areas (Figure 3B, C and Figure S6). In addition, detailed analysis of the virally injected areas demonstrated co- localization of CCL2+ and GFAP+ cells (Figure 3D). Importantly, the areas of CD3+ T-cell infiltration show CCL2 expressing cells (Figure 3E) and a positive correlation was found between the number of infiltrated CD3+ T-cells and the number of CCL2+ cells in the adenoviral injected areas of monkey brain. in the areas of injection, which were seen to express ICAM-1, VCAM (Figures S2 and S3) in contrast with intact areas (Figure S4). In addition, infiltrating lymphocytes express LFA-1 in the extravasation areas (Figure S5). Moreover, these areas of viral injection showed a particular expression of CCL2 limited to localization of CCL2 and GFAP cells (Figure 3D). Results (A) Confocal images show co-localization of CCL2-expressing cells (red) with GFAP (green) in glioma. Insert shows a detail of a single CCL2-expressing cell (red) co-localizing with GFAP (green) and counterstained with DAPI (blue). Scale bar: 30 mm. (B) Areas of infiltration of CD3+ T-cells (green) coincide with the areas of CCL2-expressing cells (red) in glioma. Confocal images show a tumorigenic area, infiltrated with CD3+ T-cells (green) in an area with numerous CCL2-expressing cells (red). The immunostaining was combined with a counterstaining with DAPI to detect the nucleus (blue). Scale bar: 50 mm. (C) Examples of characteristic CD3 infiltration in samples of glioma. CD3+ T-cells can be observed grouped in BVs (BV, limited by broken red line in 1 and 3) but also infiltrated in the parenchyma (2, 4, blue insert magnified in 5). (D) CCL2 expression correlates with the infiltration of T-cells in the tumor areas. The quantification in serial sections of the number of T-cells, either infiltrated or located in the BV lumen, revealed that the level of infiltration of CD3+ T-cells in the parenchyma (green) is positively correlated with the level of CCL2 expression (BV; BV lumen). doi:10.1371/journal.pone.0030762.g002 T-cells into two different populations: T-cells inside the BV lumen and those that were extravasated (Figure 2C). Interestingly, we found that the amount of T-cells circulating in the BV lumen was independent of CCL2 expression and no correlation was observed (Figure 2D). Conversely, a significant correlation was seen between the expression of CCL2 and the infiltration of T-cells in the tumorigenic parenchyma (Figure 2D). These results suggest that the presence of CCL2+ cells may be important for the extravasation of lymphocytes, although a chemotactic effect of secreted CCL2 into the blood circulation does not seem to contribute to this phenomenon. To investigate the role and cell type of CCL2 in other inflammatory scenarios two animal experimental models of inflammation and T-cell infiltration were studied, first using brain PLoS ONE \| www.plosone.org February 2012 \| Volume 7 \| Issue 2 \| e30762 February 2012 \| Volume 7 \| Issue 2 \| e30762 4 Astrocytes Mediate T-Cell Infiltration the injection area (Figure 3A). Importantly, confocal analysis of the infiltrated areas showed highly immunoreactive CCL2- expressing cells around BVs that were not present in intact (non- injected) areas (Figure 3B, C and Figure S6). In addition, detailed analysis of the virally injected areas demonstrated co- localization of CCL2+ and GFAP+ cells (Figure 3D). Results (E) The stereological quantification of serial adjacent sections demonstrated that the number of infiltrated CD3+ cells correlates with CCL2+ cells in adenoviral injected areas in monkey brain. doi:10.1371/journal.pone.0030762.g003 February 2012 \| Volume 7 \| Issue 2 \| e30762 PLoS ONE \| www.plosone.org 5 Astrocytes Mediate T-Cell Infiltration Since CD3+ T-cells seem to respond to CCL2 in the inflamed parenchyma in the human glioma and monkey brain, we analyze their ability to express the CCL2 receptor (CCR2) in their cell surface. We immuno-stained the glioma samples and monkey brain sections with an antibody against CCR2 combined with CD3 T-cell marker. Importantly, we observed that CD3+ T-cells are able to express CCR2 in their surface (Figure 4), which makes possible the interaction ligand-receptor in the extravasation phenomenon. In addition, to rule out whether the immunoposi- tivity for CCL2 in astrocytes might reflect the binding to CCR2, we immunostained the sections with an antibody against CCR2 combined with the astrocytic marker GFAP. We observe that CCR2 is not expressed in astrocytes (Figure S7) suggesting that the immunopositivity for CCL2 in astrocytes does not correspond to ligand binding to its receptor. infiltration is lowered with 0.1 mg/ml but it is significantly diminished with 1 mg/ml which suggests a dose dependent effect of the CCL2 neutralizing antibody administration. Taken together, these results show that CCL2 functionally mediates the extravasation of T-cells in mouse brain upon inflammation. Our results suggest that CCL2-expressing astrocytes may contribute to the specific extravasation of lymphocytes. Since there is no correlation between CCL2+ astrocytes and T-cell number in the lumen of the BV at the sites of inflammation, a chemotactic effect of secreted CCL2 does not seem to contribute to this phenomenon. Alternatively, CCL2-expressing astrocytes might need to physically interact with T-cells in order to mediate lymphocyte extravasation. To explore this possibility a detailed high-resolution confocal analysis of the anatomical location of infiltrating T-cells in the BVs and in the brain parenchyma of inflammatory areas was carried out. Importantly, specific contacts between CCL2+ cells and CD3+ lymphocytes were found in the areas of infiltration (Figure 6A and B) and at the periphery of BVs (Figure 6C), suggesting that the specific attachment of T- cells to the perivascular astrocytes may be a crucial event in the extravasation of T-cells in the inflamed brain (Figure 6D). Results g g p In order to further understand the functionality of CCL2 in mediating T-cell infiltration into the brain parenchyma, we induced brain inflammation with the stereotaxic injection of LPS in the cortex and striatum of mice. After the LPS injection, we observed a clear T-cell infiltration in the brain parenchyma evidenced by the expression of T-cell markers, CD3, CD4 and CD8 at the injection area (Figure 5A and B). The specific infiltration of T-cells in the brain after LPS was observed concomitantly with an increase in CCL2 expression limited to the injected areas. In fact, we observed a significant positive correlation between the number of infiltrated T-cells and the number of CCL2+ cells (Figure 5B and Figure S8). Detailed confocal co-localization analysis demonstrated that the CCL2+ cells were also astrocytes in LPS-induced infiltration in mice (Figure 5C), similar to the situation observed in human gliomas and monkey brain. These results suggest that CCL2-expressing astrocytes may contribute to the T-cell infiltration into the brain parenchyma. To better understand the intraparenchymal effect of CCL2, we set up an experiment using an anti-CCL2 neutralizing antibody, injected directly into the brain through an intracranial cannula as described in detail in the methods section. Briefly, after placing a guiding cannula in the mouse brain parenchyma, we waited 15 days to allow the restoration of the inflammation caused by the surgery (see diagram in Figure 5D). Then, we injected LPS through the implanted guiding cannula to induce inflammation, and after 4 days we injected either anti-CCL2 neutralizing antibody or the specific immunoglobulin isotype as a control. The histological analysis of the infiltration of CD4 and CD8 T-cells revealed that the injection of anti-CCL2 reduces significantly the infiltration of CD4+ and CD8+ lymphocytes (Figure 5D). This Discussion (D) Astrocytes express CCL2 after LPS injection in mouse brain. Confocal images of the injected areas show the co-localization of GFAP+ astrocytes (green) and CCL2 (red) in mouse brain. DAPI (blue) was used as a nuclear counterstaing. * p,0.05 ANOVA-test. doi:10.1371/journal.pone.0030762.g005 Astrocytes Mediate T-Cell Infiltration g of CCL2 attenuates the LPS-mediated T-cell infiltration. (A) Detail of CD4+ and CD8+ T-cell PS injected mice. Scale bar: 20 mm. (B) The number of infiltrated T-cells (CD4+, CD8+ and CD3+ T-cells) correlates he areas of LPS injection. (C) Intraparenchymal injection of anti-CCL2 antibodies attenuates the LPS-induced ouse brain parenchyma. The diagram on the top shows the arrangement of the experiment. Graphs show the brain parenchyma surrounding the injection site. (D) Astrocytes express CCL2 after LPS injection in mouse brain. s show the co-localization of GFAP+ astrocytes (green) and CCL2 (red) in mouse brain. DAPI (blue) was used as a OVA-test. 05 Figure 5. Intracerebral blocking of CCL2 attenuates the LPS-mediated T-cell infiltration. (A) Detail of CD4+ and CD8+ T-cell immunostaining in the striatum of LPS injected mice. Scale bar: 20 mm. (B) The number of infiltrated T-cells (CD4+, CD8+ and CD3+ T-cells) correlates with the number of CCL2+ cells in the areas of LPS injection. (C) Intraparenchymal injection of anti-CCL2 antibodies attenuates the LPS-induced infiltration of lymphocytes in the mouse brain parenchyma. The diagram on the top shows the arrangement of the experiment. Graphs show the density of CD8 and CD4 T-cells in the brain parenchyma surrounding the injection site. (D) Astrocytes express CCL2 after LPS injection in mouse brain. Confocal images of the injected areas show the co-localization of GFAP+ astrocytes (green) and CCL2 (red) in mouse brain. DAPI (blue) was used as a nuclear counterstaing. * p,0.05 ANOVA-test. doi:10.1371/journal.pone.0030762.g005 anatomical contact between lymphocytes and CCL2+ astrocytes takes place in the internal wall of BV and we suggest that may have a contributing role in the actual infiltration to the brain parenchyma. chemotactic, but our results suggest that the physical contact between T-cells and astrocytes may also contribute to the actual internalization into the brain parenchyma proper, probably after the two phenomena of rolling and adhesion, which is coherent with the induction of the uropod formation by CCL2 and penetration through the endothelium, contributing to diapedesis (Figure 6D). Our results also narrow down the controversy regarding the cell type that is responsible of CCL2 expression in the brain. Discussion In the present work, we show that, independently of the species and of the CNS inflammatory scenario, the extravasation of lymphocytes is mediated by CCL2-expressing astrocytes. In the three inflammatory situations illustrated, we found a specific increase of CCL2+ cells in the affected areas with a concomitant T-cell infiltration. Importantly, our confocal images demonstrate that perivascular astrocytes are responsible for the expression of CCL2 in the three analyzed T-cell-infiltration situations in the brain, in the three different species and with multiple antibodies specific for CCL2. Our results demonstrate that the expression of CCL2 by astrocytes contributes to the entrance of lymphocytes in the brain parenchyma. In the experiments performed in mice, the blocking of CCL2 by a specific neutralizing antibody attenuated the infiltration of lymphocytes which suggests that CCL2, expressed by astrocytes, contributes to the internalization of the T-cells into the parenchyma. Furthermore, the fact that the level of CCL2 expression correlates with the T-cell infiltration proper and not with the number of lymphocytes circulating in the BVs, strongly suggests that the presence of CCL2+ astrocytes contrib- utes to the process of lymphocyte extravasation and play a part in the cellular entrance. In addition, our data demonstrate that the Figure 4. T cells express CCL2 receptor. Expression of CCR2 in T-cells in human glioma (top panel) and monkey brain (bottom panel). CD3+ T- cells (green) express CCR2 in their surface (magenta). Nucleus is stained with DAPI (blue). doi:10.1371/journal.pone.0030762.g004 Figure 4. T cells express CCL2 receptor. Expression of CCR2 in T-cells in human glioma (top panel) and monkey brain (bottom panel). CD3+ T- cells (green) express CCR2 in their surface (magenta). Nucleus is stained with DAPI (blue). doi:10.1371/journal.pone.0030762.g004 February 2012 \| Volume 7 \| Issue 2 \| e30762 PLoS ONE \| www.plosone.org 6 Astrocytes Mediate T-Cell Infiltration Figure 5. Intracerebral blocking of CCL2 attenuates the LPS-mediated T-cell infiltration. (A) Detail of CD4+ and CD8+ T-cell immunostaining in the striatum of LPS injected mice. Scale bar: 20 mm. (B) The number of infiltrated T-cells (CD4+, CD8+ and CD3+ T-cells) correlates with the number of CCL2+ cells in the areas of LPS injection. (C) Intraparenchymal injection of anti-CCL2 antibodies attenuates the LPS-induced infiltration of lymphocytes in the mouse brain parenchyma. The diagram on the top shows the arrangement of the experiment. Graphs show the density of CD8 and CD4 T-cells in the brain parenchyma surrounding the injection site. PLoS ONE \| www.plosone.org Discussion Previous studies in biopsies of patients with multiple sclerosis and after brain injury in mice have suggested that CCL2 is expressed by astrocytes but no co-localization analysis of the proteins was conducted [11,12]. In the present work, we show that CCL2 co-localizes with GFAP in three different species, with two different antibodies, and in three different situations of inflammatory-mediated lymphocyte infiltration. (Figure 6D). From a therapeutic point of view, the manipulation of CCL2 may have beneficial effect in neurodegenerative diseases. CCL2 expression has been related with the aggressiveness of the glioma [13] and, in fact, the use of CCL2 neutralizing antibodies has been suggested as a possible strategy for their treatment [14]. However, research into CCL2-mediated infiltration has mainly focused on the role of tumor-infiltrated macrophages but not other cell types [15]. Our present work suggests that, like macrophages, the CCL2 may also mediate lymphocyte infiltration in the glioma, and contribute to the level of aggressiveness. In fact, in a previous Importantly, in our study we hypothesize that the anatomical apposition of T-cells and CCL2+ astrocytes may be an important event in the internalization of lymphocytes in the brain parenchyma. The CCL2 function was thought to be mainly February 2012 \| Volume 7 \| Issue 2 \| e30762 February 2012 \| Volume 7 \| Issue 2 \| e30762 7 d d h T ll i fil li ll d ll i h Th f h i l i Figure 6. T-cells come into contact with CCL2+ perivascular astrocytes in the areas of infiltration. (A) CCL2 is highly expressed in perivascular astrocytes where T-cells infiltrate the brain tumor. Detailed confocal analysis of samples of gliomas revealed that tumorigenic areas show CCL2-expressing astrocytes (red) with infiltration of CD3+ T-cells (green). Nuclei are stained with DAPI (blue). (B) Infiltration of T-cells occurs throughout multiple anatomical contacts between T-cells and CCL2-expressing astrocytes. Image B1 shows numerous CD3+ T-cells (green) establishing specific contacts with CCL2+ cells (red). In pictures B2, 3 and 4, a detail of T-cells (green) in contact with CCL2 perivascular astrocytes. Image B4 shows a specific detail of a CD3/CCL2 contact on the xy axis and the two lateral views on the z axis. (C) Confocal analysis of a CD3+ T-cell coming into contact with a CCL2+ cell located at the perivascular area of adenoviral injected monkey brain. The three dimensional transparency (Composite) of a stack of images shows a CD3+ T-cell (green), in close apposition to the endothelium, marked with Col-IV (red), and contacting a CCL2+ cell (magenta). A detail of the CD3-CCL2 contact is also shown in a 0.5 mm optical section on the xy axis and the two lateral views on the z axis. Acknowledgments Figure S3 VCAM-1 expression in BVs in areas of infiltration after viral injection in monkey brain. Top panel shows over-expression of VCAM-1 (red) in perivascular areas, where T-cells (green) infiltrate in the brain parenchyma. DAPI was used to stain the nuclei (blue). Bottom panel show a detail of the area of infiltration. (TIF) We thank Marı´a G. Castro and Pedro R. Lowenstein, from the Board of Governors’ Gene Therapeutics Research Institute at Cedars-Sinai Medical, Center, Los Angeles, USA, for kindly providing the adenoviral vectors used in the monkey study. We would also like thank all the personnel from SAI (Servicio de Apoyo a la Investigacio´n), for the help provided at the University of Murcia, especially Marı´a Garcı´a. The authors would also like to thank Mr. P. Thomas for comments and language suggestions on this manuscript. Figure S4 Intact areas do not express ICAM-1 or VCAM- 1 in monkey brain. Confocal images of BV in intact areas of the macaque brain. ICAM-1 and VCAM-1 are not over-expressed in BV and no CD3+ T-cell infiltration is seen in non injected areas. (TIF) 9. Barcia C, Jimenez-Dalmaroni M, Kroeger KM, Puntel M, Rapaport AJ, et al. (2007) One-year expression from high-capacity adenoviral vectors in the brains of animals with pre-existing anti-adenoviral immunity: clinical implications. Mol Ther 15: 2154–2163. (TIF) Figure S2 ICAM-1 expression in BVs in areas of infiltration after viral injection in monkey brain. Top panel shows over-expression of ICAM-1 (red) in perivascular areas, where T-cells (green) infiltrate the brain parenchyma. DAPI was used to stain the nuclei (blue). Bottom panel shows different putative steps (from 1 to 6) of T-cell infiltration in the brain. (1, 2) T-cells (green) rolling through the ICAM-1+ endothelial wall (red). Picture 2 shows a rolling T-cell displaying a putative uropod (white arrow). (3, 4) T-cells (green) in the adhesion process. (5, 6) T-cells in the extravasation process. (TIF) Figure S8 Increase in the number of CCL2+ cells in the mouse brain after LPS intrastriatal injection. (A) Repre- sentative picture of CCL2+ cells in the mouse striatum after LPS injection compared to a saline injection. (B) Quantification of CCL2+ cells in the injected striatum in a group of mice injected with saline compared with a group of mice injected with LPS. A dramatic increase of CCL2+ cells can be observed in the LPS injected mice. p,0.05 Student t-test. (TIF) (Figure 6D). This suggests that the most tumor-infiltrated lymphocytes are regulatory, reducing the activity of the cytolitic PLoS ONE \| www.plosone.org February 2012 \| Volume 7 \| Issue 2 \| e30762 February 2012 \| Volume 7 \| Issue 2 \| e30762 8 Astrocytes Mediate T-Cell Infiltration trauma, stroke, as well as during chronic affections like multiple sclerosis or Alzheimer’s disease, prolonged and sustained inflam- mation mediated by CCL2 may have cytotoxic effects, aggravating the incidence and the severity of the disease [16]. Therefore, targeting CCL2, more specifically in perivascular astrocytes, in CNS inflammatory scenarios may be particularly important from a therapeutic perspective. parenchyma. DAPI was used to stain the nuclei (blue). Bottom panel shows a detail of the area of infiltration of T and B lymphocytes co-localizing with LFA-1. (TIF) Figure S6 Staining of collagen-IV was performed to unequivocally delineate blood vessels shown in figure 3B. Confocal images show BVs in monkey brain sections stained with DAPI (white) and collagen-IV (green). In the merged images DAPI is shown in blue. (TIF) (Figure 6D). (D) Diagram showing how T-cells may come into contact with CCL2-expressing astrocytes in the edge of BVs suggesting that CCL2+ astrocytes contribute to the extravasation of T-cells in the brain parenchyma. doi:10.1371/journal.pone.0030762.g006 Astrocytes Mediate T-Cell Infiltration Astrocytes Mediate T-Cell Infiltration Figure 6. T-cells come into contact with CCL2+ perivascular astrocytes in the areas of infiltration. (A) CCL2 is highly expressed in perivascular astrocytes where T-cells infiltrate the brain tumor. Detailed confocal analysis of samples of gliomas revealed that tumorigenic areas show CCL2-expressing astrocytes (red) with infiltration of CD3+ T-cells (green). Nuclei are stained with DAPI (blue). (B) Infiltration of T-cells occurs throughout multiple anatomical contacts between T-cells and CCL2-expressing astrocytes. Image B1 shows numerous CD3+ T-cells (green) establishing specific contacts with CCL2+ cells (red). In pictures B2, 3 and 4, a detail of T-cells (green) in contact with CCL2 perivascular astrocytes. Image B4 shows a specific detail of a CD3/CCL2 contact on the xy axis and the two lateral views on the z axis. (C) Confocal analysis of a CD3+ T-cell coming into contact with a CCL2+ cell located at the perivascular area of adenoviral injected monkey brain. The three dimensional transparency (Composite) of a stack of images shows a CD3+ T-cell (green), in close apposition to the endothelium, marked with Col-IV (red), and contacting a CCL2+ cell (magenta). A detail of the CD3-CCL2 contact is also shown in a 0.5 mm optical section on the xy axis and the two lateral views on the z axis. (D) Diagram showing how T-cells may come into contact with CCL2-expressing astrocytes in the edge of BVs suggesting that CCL2+ astrocytes contribute to the extravasation of T-cells in the brain parenchyma. doi:10.1371/journal.pone.0030762.g006 cells and allowing the tumor to grow. Therefore, the manipulation of CCL2 may be one of the particular therapeutic targets to control the infiltration of lymphocytes in brain tumors. Accord- ingly, in other neuroinflammatory processes, such as acute brain report, we demonstrated that T-cells infiltrate gliomas to contact tumorigenic cells although a very low percentage shows cytolitic features [6]. Author Contributions Conceived and designed the experiments: C. Barcia Sr. C. Barcia Jr. MTH. Performed the experiments: MAC AG CMR FR EDM AP JMG EFV C. Barcia Sr. C. Barcia Jr. MTH. Analyzed the data: MAC AG CMR C. Barcia Sr. C. Barcia Jr. MTH. Contributed reagents/materials/analysis tools: MAC AG CMR FR AP JMG EF C. Barcia Sr. C. Barcia Jr. MTH. Wrote the paper: MAC C. Barcia Jr. MTH. Conceived and designed the experiments: C. Barcia Sr. C. Barcia Jr. MTH. Performed the experiments: MAC AG CMR FR EDM AP JMG EFV C. Barcia Sr. C. Barcia Jr. MTH. Analyzed the data: MAC AG CMR C. Barcia Sr. C. Barcia Jr. MTH. Contributed reagents/materials/analysis tools: MAC AG CMR FR AP JMG EF C. Barcia Sr. C. Barcia Jr. MTH. Wrote the paper: MAC C. Barcia Jr. MTH. Figure S5 LFA-1 expression in lymphocytes in areas of infiltration after viral injection in monkey brain. Top panel shows over-expression of LFA-1 (magenta) in perivascular areas, where T-cells (green) and B cells (red) infiltrate the brain 8. Zirger JM, Liu C, Barcia C, Castro MG, Lowenstein PR (2006) Immune regulation of transgene expression in the brain: B cells regulate an early phase of elimination of transgene expression from adenoviral vectors. Viral Immunol 19: 508–517. 7. Barcia C, Gomez A, de Pablos V, Fernandez-Villalba E, Liu C, et al. (2008) CD20, CD3, and CD40 ligand microclusters segregate three-dimensionally in vivo at B-cell-T-cell immunological synapses after viral immunity in primate brain. J Virol 82: 9978–9993. 6. Barcia C, Jr., Gomez A, Gallego-Sanchez JM, Perez-Valles A, Castro MG, et al. (2009) Infiltrating CTLs in human glioblastoma establish immunological synapses with tumorigenic cells. Am J Pathol 175: 786–798. Supporting Information Figure S1 Tumors show typical anatomopathological glioma characteristics. (A) Area of a sample of glioma with hypercellular appearance with hemorragic areas. Insert shows details of the marked hypercellularity. (B) Sample of a case of glioma showing pleomorphic cells, aberrant mitosis and areas of necrosis. Insert shows a detail of the pleomorhic cells and mitosis. Necrosis area is indicated with an asterisk (). (C) Sample of glioma showing glomeruloid vessels (), areas of necrosis (arrow) and gemistocytic cells (Insert). (D) Sample of glioma showing glomeruloid vessels (). Insert shows a detail of a glomeruloid vessel. Scale bars are indicated in each insert. (TIF) Figure S7 CCR2 is not expressed in GFAP+ astrocytes. Immuno-staining of CCR2 in monkey brain and samples of glioma combined with the astrocytic marker GFAP. (A) The areas of adenoviral injection in monkey brain show CCR2+ cells (magenta) and do not co-localize with GFAP marker (green). DAPI was used as a counterstaining (blue). Insert shows contact between a CCR2+ cell and a GFAP+ astrocyte. (B) Samples of glioma (GBM1, GBM2 and GBM3) show CCR2+ cells (magenta) and do not co-localize with GFAP+ astrocytes. DAPI was used as a counterstaining (blue). Scale bars: 50 mm. 10. Thomas CE, Schiedner G, Kochanek S, Castro MG, Lowenstein PR (2000) Peripheral infection with adenovirus causes unexpected long-term brain inflammation in animals injected intracranially with first-generation, but not with high-capacity, adenovirus vectors: toward realistic long-term neurological gene therapy for chronic diseases. Proc Natl Acad Sci U S A 97: 7482–7487. g py 11. Van Der Voorn P, Tekstra J, Beelen RH, Tensen CP, Van Der Valk P, et al. (1999) Expression of MCP-1 by reactive astrocytes in demyelinating multiple sclerosis lesions. Am J Pathol 154: 45–51. 12. Glabinski AR, Balasingam V, Tani M, Kunkel SL, Strieter RM, et al. (1996) Chemokine monocyte chemoattractant protein-1 is expressed by astrocytes after mechanical injury to the brain. J Immunol 156: 4363–4368. 13. Platten M, Kretz A, Naumann U, Aulwurm S, Egashira K, et al. (2003) Monocyte chemoattractant protein-1 increases microglial infiltration and aggressiveness of gliomas. Ann Neurol 54: 388–392. gg g 14. Zhu X, Fujita M, Snyder LA, Okada H (2010) Systemic delivery of neutralizing antibody targeting CCL2 for glioma therapy. J Neurooncol. 16. Conductier G, Blondeau N, Guyon A, Nahon JL, Rovere C (2010) The role of monocyte chemoattractant protein MCP1/CCL2 in neuroinflammatory diseases. J Neuroimmunol 224: 93–100. 15. Desbaillets I, Tada M, de Tribolet N, Diserens AC, Hamou MF, et al. (1994) Human astrocytomas and glioblastomas express monocyte chemoattractant protein-1 (MCP-1) in vivo and in vitro. Int J Cancer 58: 240–247. References 1. del Pozo MA, Cabanas C, Montoya MC, Ager A, Sanchez-Mateos P, et al. (1997) ICAMs redistributed by chemokines to cellular uropods as a mechanism for recruitment of T lymphocytes. J Cell Biol 137: 493–508. 6. Barcia C, Jr., Gomez A, Gallego-Sanchez JM, Perez-Valles A, Castro MG, et al. (2009) Infiltrating CTLs in human glioblastoma establish immunological synapses with tumorigenic cells. Am J Pathol 175: 786–798. y p g 7. Barcia C, Gomez A, de Pablos V, Fernandez-Villalba E, Liu C, et al. (2008) CD20, CD3, and CD40 ligand microclusters segregate three-dimensionally in vivo at B-cell-T-cell immunological synapses after viral immunity in primate brain. J Virol 82: 9978–9993. 2. del Pozo MA, Sanchez-Mateos P, Nieto M, Sanchez-Madrid F (1995) Chemokines regulate cellular polarization and adhesion receptor redistribution during lymphocyte interaction with endothelium and extracellular matrix. Involvement of cAMP signaling pathway. J Cell Biol 131: 495–508. 3. Sanchez-Madrid F, Serrador JM (2009) Bringing up the rear: defining the roles of the uropod. Nat Rev Mol Cell Biol 10: 353–359. 4. Oppenheim JJ, Zachariae CO, Mukaida N, Matsushima K (1991) Properties of the novel proinflammatory supergene ‘‘intercrine’’ cytokine family. Annu Rev Immunol 9: 617–648. 5. Deshmane SL, Kremlev S, Amini S, Sawaya BE (2009) Monocyte chemoat- tractant protein-1 (MCP-1): an overview. J Interferon Cytokine Res 29: 313–326. 5. Deshmane SL, Kremlev S, Amini S, Sawaya BE (2009) Monocyte chemoat- tractant protein-1 (MCP-1): an overview. J Interferon Cytokine Res 29: 313–326. PLoS ONE \| www.plosone.org February 2012 \| Volume 7 \| Issue 2 \| e30762 9 Astrocytes Mediate T-Cell Infiltration Astrocytes Mediate T-Cell Infiltration Astrocytes Mediate T-Cell Infiltration PLoS ONE \| www.plosone.org February 2012 \| Volume 7 \| Issue 2 \| e30762 10
https://openalex.org/W4389571640	https://www.qeios.com/read/0032QB/pdf	English	null	Review of: "Exploring the ATP Synthesis in Unique Cellular Structures: A Preliminary Hypothesis"	null	2,023	cc-by	294	Qeios, CC-BY 4.0 · Review, December 11, 2023 Qeios ID: 0032QB · https://doi.org/10.32388/0032QB Review of: "Exploring the ATP Synthesis in Unique Cellular Structures: A Preliminary Hypothesis" Silvana Pinna1 1 University of Strasbourg Silvana Pinna1 Potential competing interests: No potential competing interests to declare. The hypothesis discussed in the manuscript on ATP synthesis through the SET is quite interesting, and the paper is well- organised. I saw the changes made from v1 and they are appropriate, however there are still a few points that are not clear to the reader. Because this is the proposal of a new mechanism, I believe that a more thorough literature review on the existing ideas would be beneficial to put the new idea into context. The way the data is presented could be clearer, and it might be useful to discuss potentially confounding factors and controls more in detail, especially the way the different results relate to each other. In my view, more experimental evidence is needed in order to draw the conclusions the manuscript presents. For example, how is the ATP yield confirmed to be solely due to the SET and not from the other well-known processes? Or how is it isolated and preserved from consumption from metabolic processes? It may be something that has been done in the experimental protocol, but it needs to be reported for the reader to come to the same conclusions. I am also struggling to understand the advantage of using labelled H2PO4- . On a minor note, I was unable to find the definition of ‘AA’ in the text. Altogether the hypothesis introduces interesting concepts, and I believe that once comments are addressed, the article would be in really good shape. Qeios ID: 0032QB · https://doi.org/10.32388/0032QB Qeios ID: 0032QB · https://doi.org/10.32388/0032QB 1/1
https://openalex.org/W2910933284	https://www.matec-conferences.org/10.1051/matecconf/201925405012/pdf	English	null	Testing of a force sensor used to measure the briquetting process parameters of lignocellulosic materials	MATEC web of conferences	2,019	cc-by	2,627	Testing of a force sensor used to measure the briquetting process parameters of lignocellulosic materials Krzysztof Talaśka1,, Dominik Wojtkowiak1 , Ireneusz Malujda1 and Krzysztof Wałęsa1 1 Chair of Basics of Machine Design, Poznan University of Technology, Poznan, 60-965, Poland Krzysztof Talaśka1,, Dominik Wojtkowiak1 , Ireneusz Malujda1 and Krzysztof Wałęsa1 1 Chair of Basics of Machine Design, Poznan University of Technology, Poznan, 60-965, Poland Abstract. The study presents a control and measurement system for the drive parameters of screw compactor machine with open working chamber for shredded materials. The study demonstrates the construction and working principle of the force sensor for measuring the axial force at the compacting screw. The calibration method for the measuring system was presented together with example measurement results for compaction of broken up rye straw, oat straw and hay. The study results were analyzed for feasibility of use for the compaction of materials with specific thermomechanical properties. The conclusion presents the possible forms of application of the established solution in mass production. Keywords: worm/screw-based compaction machine, force sensor MATEC Web of Conferences 254, 05012 (2019) MMS 2018 MATEC Web of Conferences 254, 05012 (2019) MMS 2018 https://doi.org/10.1051/matecconf/201925405012 1 Introduction Manufacturing of solid fuels from broken down lignocellulosic materials has been steadily growing in popularity in the last years. This is caused by overproduction of materials suitable as fuel. Such overproduction is also observed in Poland since many years. Table 1 presents the balance of the total production compared to the utilization together with the forecast for the years 2010-2030. Table 1. The balance of the total production and utilization of straw in the years 2010-2030 [1-8] Table 1. The balance of the total production and utilization of straw in the years 2010-2030 [1-8] Year 2010 2011 2012 2013 2014 Forecast 2020 2030 Production [million tons] 29.753 28.89 30.793 29.343 35.608 30.533 30.862 Utilization [million tons] Mulch 11.843 11.24 10.698 10.303 10.47 9.868 9.319 Fodder 3.923 3.914 3.898 3.763 3.806 3.527 3.221 Green manure 3 3 3 3 3 3 3 Bedding for mycelium 1 1 1 1 1 1 1 Balance 9.987 9.736 12.198 11.277 17.322 13.138 14.322 Table 1. The balance of the total production and utilization of straw in the years 2010-2030 [1-8] * Corresponding author: krzysztof.talaska@put.poznan.pl Reviewers: Grzegorz Domek, Andrzej Kołodziej © 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/). MATEC Web of Conferences 254, 05012 (2019) MMS 2018 https://doi.org/10.1051/matecconf/201925405012 Compaction of materials enables the change of their mechanical and usable properties as well as transport and storage conditions [8-15]. The manufacturing of solid fuels calls for the utilization of proper compaction technique. The most popular are piston, roller and screw based solutions. The utilization of any of the above methods depends on proper preconditioning of the material to be compacted. Pelletization utilizing roller technology calls for a finer breakdown of straw material. Whereas worm/screw-based compaction allows to compact cut straw with length from several to several dozen millimeters [8-15]. Fig. 1 shows the diagram of a compacting machine utilizing the screw technique with open forming sleeve. Fig. 1. Worm-based compaction machine with open forming sleeve used in the manufacturing of briquette from broken down lignocellulosic materials: 1 – drive motor, 2 – mechanical transmission, 3 – compacted material, 4 – worm, 5 – heated forming (compacting) sleeve, 6 – final geometric form of the agglomerate [8] 1 2 3 4 5 6 Fig. 1. Worm-based compaction machine with open forming sleeve used in the manufacturing of briquette from broken down lignocellulosic materials: 1 – drive motor, 2 – mechanical transmission, 3 – compacted material, 4 – worm, 5 – heated forming (compacting) sleeve, 6 – final geometric form of the agglomerate [8] 2 Process parameters for the compaction of lignocellulosic materials utilizing compacting screw technique Hydraulic rotational force gauge: 1 – shaft, 2 – body, 3 – force gauge body 1, 4 – force gauge body 2, 5 – sealing ring, 6 – pressure chamber, 7 – threaded sleeve, 8, 9, 10 – bearing, 11 – chain wheels, 12 – slot for pressure gauge, 13 – slot with inlet groove for the installation of worm pin, Fa – axial force [8, 16] The operating principle of the presented force gauge is based on the direct measurement of the axial force based on the pressure change in the hydraulic chamber (between items no. 2, 3 and 4). After calibrating the force gauge using a durometer, it can be used to measure axial force for compacting selected materials. The calibration entails identifying the force- pressure characteristics allowing to use the pressure value within the hydraulic chamber to calculate the axial force accounting for resistances from construction. The operating principle of the presented force gauge is based on the direct measurement of the axial force based on the pressure change in the hydraulic chamber (between items no. 2, 3 and 4). After calibrating the force gauge using a durometer, it can be used to measure axial force for compacting selected materials. The calibration entails identifying the force- pressure characteristics allowing to use the pressure value within the hydraulic chamber to calculate the axial force accounting for resistances from construction. 2 Process parameters for the compaction of lignocellulosic materials utilizing compacting screw technique The working system of the machine utilizing the compacting screw consists of the two basic components: worm screw set in rotational motion via electrical drive together with the forming sleeve in which the worm performs the rotating motion. Fig. 2 presents the working system diagram of the machine utilizing compacting screw. Fig. 2. Geometric properties of the working system of the compaction machine for broken down lignocellulosic materials (compacting screw technique): D – diameter of the cylindrical part of the worm, dr – worm root diameter, P – worm pitch, Fre – resistance force, Fa – axial force, Tr – torque, A – main compaction area of the material dr D P Tr Fa Fre A Fig. 2. Geometric properties of the working system of the compaction machine for broken down lignocellulosic materials (compacting screw technique): D – diameter of the cylindrical part of the worm, dr – worm root diameter, P – worm pitch, Fre – resistance force, Fa – axial force, Tr – torque, A – main compaction area of the material 2 2 MATEC Web of Conferences 254, 05012 (2019) MMS 2018 https://doi.org/10.1051/matecconf/201925405012 Analyzing the operating principle of such a working system, we can identify two basic functional parameters: the torque necessary to affect the rotating motion for compaction Tr, and resistance force Fa, which puts load on the worm during machine operation. The value of both parameters depend partially on the thermomechanical properties of the compacted materials. Therefore, during operation of the compacting screw mechanism, process temperature is another parameter to materially affect the entire process. Identifying the torque and axial force values may be critical for machine control to facilitate efficient compaction process. Therefore, an attempt was made to suggest a construction solution for a specialized force gauge dedicated for this type of machine. The proposed solution is protected by a Polish patent no. 225645. Fig. 3 demonstrates the construction diagram of the force gauge. Fig. 3. Hydraulic rotational force gauge: 1 – shaft, 2 – body, 3 – force gauge body 1, 4 – force gauge body 2, 5 – sealing ring, 6 – pressure chamber, 7 – threaded sleeve, 8, 9, 10 – bearing, 11 – chain wheels, 12 – slot for pressure gauge, 13 – slot with inlet groove for the installation of worm pin, Fa – axial force [8, 16] Fig. 3. 3 Methodology and scope of test measurements The examination was carried out on a compaction machine utilizing compaction worm screw. The compaction process was undertaken for oat straw, rye straw and hay (Fig. 4). The examination was carried out for three rotation speeds of the worm: 210, 280 and 350 rpm. Figs. 5-7 present example results of the examination. Table 2 presents a breakdown of collected results of the axial force examination for the three materials at three rotation speeds of the worm. 3 3 https://doi.org/10.1051/matecconf/201925405012 MATEC Web of Conferences 254, 05012 (2019) MMS 2018 Fig. 4. Materials used for the examination: oat straw, rye straw, hay Fig. 4. Materials used for the examination: oat straw, rye straw, hay 0 1 2 3 4 5 6 0 20 40 60 80 100 120 140 Axial force Fa [kN] Time [s] 210 rpm 280 rpm 350 rpm Fig. 5. The change of axial force Fa as a function of time; rye straw 0 1 2 3 4 5 6 0 20 40 60 80 100 120 140 Axial force Fa [kN] Time [s] 210 rpm 280 rpm 350 rpm Fig. 6. The change of axial force Fa as a function of time; oat straw Fig. 4. Materials used for the examination: oat straw, rye straw, hay 0 1 2 3 4 5 6 0 20 40 60 80 100 120 140 Axial force Fa [kN] Time [s] 210 rpm 280 rpm 350 rpm Fig. 5. The change of axial force Fa as a function of time; rye straw 0 1 2 3 4 5 6 0 20 40 60 80 100 120 140 Axial force Fa [kN] Time [s] 210 rpm 280 rpm 350 rpm i h h f i l f f i f i Fig. 5. The change of axial force Fa as a function of time; rye straw 0 1 2 3 4 5 6 0 20 40 60 80 100 120 140 Axial force Fa [kN] Time [s] 210 rpm 280 rpm 350 rpm Fig. 6. The change of axial force Fa as a function of time; oat straw 0 1 2 3 4 5 6 0 20 40 60 80 100 120 140 Axial force Fa [kN] Time [s] 210 rpm 280 rpm 350 rpm Fig. 6. The change of axial force Fa as a function of time; oat straw Fig. 6. 3 Methodology and scope of test measurements The change of axial force Fa as a function of time; oat straw 4 MATEC Web of Conferences 254, 05012 (2019) MMS 2018 https://doi.org/10.1051/matecconf/201925405012 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 0 20 40 60 80 100 120 140 Axial force Fa [kN] Time [s] 210 rpm 280 rpm 350 rpm Fig. 7. The change of axial force Fa as a function of time; hay Table 2. Breakdown of results of the examination of axial force values Fa Material Axial force Fa [kN] nś1 nś2 nś3 Rye straw 2.8-4.8 3.2-4.2 3.1-4.8 Oat straw 2.3-3.4 2.7-3.9 3.4-4.7 Hay 1.7-4.0 1.8-3.3 1.8-3.6 4 Conclusion 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 0 20 40 60 80 100 120 140 Axial force Fa [kN] Time [s] 210 rpm 280 rpm 350 rpm Fig. 7. The change of axial force Fa as a function of time; hay 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 0 20 40 60 80 100 120 140 Axial force Fa [kN] Time [s] 210 rpm 280 rpm 350 rpm Fig. 7. The change of axial force Fa as a function of time; hay Fig. 7. The change of axial force Fa as a function of time; hay Table 2. Breakdown of results of the examination of axial force values Fa Table 2. Breakdown of results of the examination of axial force values Fa Table 2. Breakdown of results of the examination of axial force values Fa Material Axial force Fa [kN] nś1 nś2 nś3 Rye straw 2.8-4.8 3.2-4.2 3.1-4.8 Oat straw 2.3-3.4 2.7-3.9 3.4-4.7 Hay 1.7-4.0 1.8-3.3 1.8-3.6 Material Axial force Fa [kN] nś1 nś2 nś3 Rye straw 2.8-4.8 3.2-4.2 3.1-4.8 Oat straw 2.3-3.4 2.7-3.9 3.4-4.7 Hay 1.7-4.0 1.8-3.3 1.8-3.6 4 Conclusion The proposed hydraulic force gauge intended for use in the compacting machine utilizing worm screw technique fulfills its purpose successfully. The variability of the compaction process of the broken down lignocellulosic materials prevent designing dedicated working systems for specific materials. It is therefore much more advantageous to utilize dedicated measurement systems allowing to monitor selected parameters during machine operation. This may be utilized to control the machine utilizing the known value of axial force load on the auger. Analyzing the results of the study, we can conclude that even within a single type of material, the variability of the axial force value is considerable. The value ranges of axial force for different materials overlap. This confirms the thesis regarding the suitability of designing dedicated, specialized measuring systems allowing constant control of the selected parameter during machine operation. References 1. GUS: Characteristics of farms in 2013 (in Polish), Warszawa (2014) 2. GUS: Production of agricultural and horticultural crops in 2014 (in Polish), Warszawa (2015) 3. GUS: Land and sown area use in 2014 (in Polish), Warszawa (2015) 4. GUS: Livestock in 2014 (in Polish), Warszawa (2015) 5. A. Faber, J. Kuś, Alternative directions of production of Polish agriculture (in Polish), Pamiętnik Puławski, 132, pp. 59-73 (2003) 6. Cz. Maćkowiak, Straw as a fertilizer on a farm without inventory (in Polish), Wieś Jutra, 5, pp. 46-48 (1998) 1. GUS: Characteristics of farms in 2013 (in Polish), Warszawa (2014) 1. GUS: Characteristics of farms in 2013 (in Polish), Warszawa (2014) 2. GUS: Production of agricultural and horticultural crops in 2014 (in Polish), Warszawa (2015) 2. GUS: Production of agricultural and horticultural crops in 2014 (in Polish), Warszawa (2015) 3. GUS: Land and sown area use in 2014 (in Polish), Warszawa (2015) 3. GUS: Land and sown area use in 2014 (in Polish), Warszawa (2015) 4. GUS: Livestock in 2014 (in Polish), Warszawa (2015) 5. A. Faber, J. Kuś, Alternative directions of production of Polish agriculture (in Polish), Pamiętnik Puławski, 132, pp. 59-73 (2003) 6. Cz. Maćkowiak, Straw as a fertilizer on a farm without inventory (in Polish), Wieś Jutra, 5, pp. 46-48 (1998) 5 5 https://doi.org/10.1051/matecconf/201925405012 MATEC Web of Conferences 254, 05012 (2019) MMS 2018 7. A. Madej, Straw balance in Poland in 2010-2014 and forecast by 2030 (in Polish), Stowarzyszenie Ekonomistów Rolnictwa i Agrobiznesu, Roczniki Naukowe, t. XVIII, z. 1, pp. 163-168 (2016) 7. A. Madej, Straw balance in Poland in 2010-2014 and forecast by 2030 (in Polish), Stowarzyszenie Ekonomistów Rolnictwa i Agrobiznesu, Roczniki Naukowe, t. XVIII, z. 1, pp. 163-168 (2016) 8. K. Talaśka, Study of research and modelling of compaction processes of powder and shredded materials (in Polish), Wydawnictwo Politechniki Poznańskiej, Poznań (2018) 9. K. Talaśka, I. Malujda, D. Wilczyński, Agglomeration of natural fibrous materials in perpetual screw technique – a challenge for designer, Procedia Engineering, 136, pp. 63-69 (2016). 10. D. Wilczyński, K. Talaśka, I. Malujda, R. Długi, The Study of Mechanical Properties of Natural Polymers in the Compacting Process, Procedia Engineering, 177, pp. 411- 418 (2017) 11. I. Malujda, D. Wilczyński, Mechanical Properties Investigation of Natural Polymers, Procedia Engineering, 136, pp. 263-268 (2016) 12. D. Wilczyński, K. Talaśka, I. Malujda, P. References Jankowiak, Experimental research on biomass cutting process, MATEC Web of Conferences, 157, Article Number 07016 (2018) 13. J. Górecki, I. Malujda, K. Talaśka, M. Kukla, P. Tarkowski, Influence of the compression length on the ultimate stress in the process of mechanical agglomeration of dry ice, Procedia Engineering, 177, pp. 363-368 (2017) 14. J. Górecki, I. Malujda, K. Talaśka, P. Tarkowski, M. Kukla, Influence of the value of limit densification stress on the quality of the pellets during the agglomeration process of CO2, Procedia Engineering, 136, pp. 269-274 (2016) 15. J. Górecki, I. Malujda, K. Talaśka, D. 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https://openalex.org/W2042205083	https://www.biodiversitylibrary.org/partpdf/183865	English	null	Young Larvae of Veromessor Pergandei (Hymenoptera: Formicidae: Myrmicinae)	Psyche	1,987	public-domain	1,717	YOUNG LARVAE OF VEROMESSOR PERGANDEI (HYMENOPTERA: FORMICIDAE: MYRMICINAE) Time was when it seemed every myrmecologist wanted to work on Veromessor pergandei, but we can find no mention of it in the last eight years of Zoological Record. When we lived with it in Death Valley and southern Nevada it became one of our favorite ants. To differentiate instars we would like the following specimens: a first instar inside an egg; a second instar inside a first instar that is ready to moult; a third instar inside a second ready to moult; etc.; a mature larva; a prepupa. Fortunately our V. pergandei material meets all the requirements, except the first. V. pergandei is poly- morphic, which presents another problem: when does subcaste dif- ferentiation begin? How can one tell whether a small larva is the young of a major or a mature of a minim; or whether a medium- sized larva is the mature of an intermediate worker or the half- grown larva of a major? In V. pergandei subcaste differences apparently begin in the fourth instar and are manifested only in size. V. pergandei presents another problem: there are two body shapes for mature worker larvae. We have no explanation for this. ♦Mailing address: 3358 NE 58th Avenue, Silver Springs, Florida 32688 Manuscript received by the editor June 4, 1987 Vermessor pergandei (Mayr) Figures 1-6. Egg. Figure 1. About 0.32 X 0.52 mm. First Instar. Figure 2. Length (through spiracles) about 0.48 mm. Entire larva feebly sclerotized. Body sac-like; head on anterior end and greater in diameter then thoracic somites. Spiracles about 0.006 mm in diameter. No spinules nor hairs on body. Cranium subcircular in anterior view. Antennae represented by 2 sensilla. About 20 head hairs, 0.013-0.025 mm long, unbranched. Mouth parts small. Labrum with a ventrolateral swelling on each half of anterior surface; 2 or 3 sensilla on each half of ventral surface. Mandibles subtriangular; apical tooth straight and sharp-pointed; 303 304 Psyche [Vol. 94 Figures 1-5. Left mandible in anterior view, XI 85; larva in side view and egg, XI 9; head in anterior view, X76. 1. Egg. 2. First Instar. 2a, Mandible; 2b, profile; 2c, head. 3. Second Instar. 3a, Mandible; 3b, profile; 3c, head. 4. Third instar. 4a, Mandible; 4b, profile; 4c, head. 5. Fourth Instar. 5a, Mandible; 5b, profiles of two sizes of larvae; 5c, head. 304 Psyche Psyche [Vol. 94 [Vol. 94 Figures 1-5. Left mandible in anterior view, XI 85; larva in side view and egg, XI 9; head in anterior view, X76. 1. Egg. 2. First Instar. 2a, Mandible; 2b, profile; 2c, head. 3. Second Instar. 3a, Mandible; 3b, profile; 3c, head. 4. Third instar. 4a, Mandible; 4b, profile; 4c, head. 5. Fourth Instar. 5a, Mandible; 5b, profiles of two sizes of larvae; 5c, head. Figures 1-5. Left mandible in anterior view, XI 85; larva in side view and egg, XI 9; head in anterior view, X76. 1. Egg. 2. First Instar. 2a, Mandible; 2b, profile; 2c, head. 3. Second Instar. 3a, Mandible; 3b, profile; 3c, head. 4. Third instar. 4a, Mandible; 4b, profile; 4c, head. 5. Fourth Instar. 5a, Mandible; 5b, profiles of two sizes of larvae; 5c, head. Figures 1-5. Left mandible in anterior view, XI 85; larva in side view and egg, XI 9; head in anterior view, X76. 1. Egg. 2. First Instar. 2a, Mandible; 2b, profile; 2c, head. 3. Second Instar. 3a, Mandible; 3b, profile; 3c, head. 4. Third instar. 4a, Mandible; 4b, profile; 4c, head. 5. Fourth Instar. 5a, Mandible; 5b, profiles of two sizes of larvae; 5c, head. medial border with a small projection. Maxillary palp with 5 sensilla on a slight elevation; galea represented by 2 sensilla. Labium short and wide; palp represented by 5 (?) sensilla. Second Instar. Vermessor pergandei (Mayr) Figures 1-6. Figure 3. Length (through spiracles) about 1.6 mm. Dorsal profile of body feebly C-shaped, ventral feebly sigmoid; T1-T3 nearly same diameter; AIII-AIV widest, tapering slightly to anterior end and more rapidly to posterior end; head and anus ventral; anus with a small posterior lip. Spiracle diameter about 0.008 mm. Integument with minute spinules in short rows on venter of thorax and all surfaces of A VIII- AX. Body hairs on T1 only; 0.006-0.018 mm long, slightly curved and with frayed tip. Cranium oval, narrowed ventrally; length subequal to width. Antennae with 3 sensilla each. Head hairs few (about 35); 0.006-0.018 mm long, with Wheeler & Wheeler — Larvae of Veromessor 305 1987] short frayed tip. Labrum feebly bilobed; anterior surface of each lobe with a few sensilla near and on ventral surface. Mandible feebly sclerotized. Maxillary palp represented by a cluster of 5 sensilla; galea a slight elevation with 2 sensilla. Labial palp represented by a cluster of 5 sensilla. Third Instar. Figure 4. Length (through spiracles) 2. 4-2. 8 mm. Similar to second instar except as follows: Body widest at AIII, venter nearly straight; dorsal profile long and C-shaped. Spiracles about 0.019 mm in diameter. Entire integument with minute spi- nules in short arcuate rows. Body hairs very few; on thorax only; 0.013-0.028 mm long, with very short-bifid tip. Cranium subhex- agonal in anterior view; width and length subequal. About 30 head hairs; 0.013-0.058 mm long, with short 2- or 3-branched tip. Labrum bilobed; each lobe with 2 sensilla on anterior surface; ven- tral surface with 3 sensilla on each half; posterior surface with 3 sensilla near ventral border of each lobe and with a cluster of 3 sensilla near middle; with small patches of isolated spinules dorsally and near middle. Maxilla with round-pointed apex and with a few short rows of minute spinules; palp a short frustum with 5 sensilla; galea a low rounded knob with 2 sensilla. Labium with small patches of spinules dorsally near middle, the spinules isolated or in short rows; palp a slightly elevated cluster of 5 sensilla; an isolated sensillum between each palp and the opening of the sericteries; the latter a short transverse slit. Hypopharynx with a few minute spi- nules in short transverse rows. Fourth Instar. Figure 5. Length (through spiracles) about 3.0-4.9 mm. Vermessor pergandei (Mayr) Figures 1-6. Similar to third instar except as follows: Body hairs sparse, generally distributed; 0.013-0.1 mm long; with slightly curved shaft and short-frayed tip. About 45 head hairs; 0.012-0.05 mm long. Posterior surface of labrum with rather coarse spinules, isolated or in short rows dorsally. Mandible moderately sclerotized; apical tooth long, narrow and sharp-pointed; subapical tooth short- er and less pointed. Maxillary palp and galea more elevated. Labial palp a low knob with 5 sensilla. Hypopharynx with numerous short transverse rows of minute spinules; upper portion with numerous short ridges [furrows?] converging into pharynx. Fifth Instar (Mature Larva). Figure 6. Length (through spira- cles) 4. 3-6. 8 mm. Similar to fourth instar except as follows: Body profile pogonomyrmecoid but of two different shapes: (1) stout, with All and AIII swollen dorsally; (2) abdomen swollen and sac- 306 Psyche [Voi. 94 Figures 6-7. Fifth Instar (Mature Larva). 6a (hairs omitted) and 6b, profiles, XI 9; 6c, left mandible in anterior view, XI 85; 6d, head in anterior view, X76. 7. Profiles of mature larvae to show size range (hairs omitted), X8. 7a, Major worker; 7b, minor worker. Psyche 306 94 94 Figures 6-7. Fifth Instar (Mature Larva). 6a (hairs omitted) and 6b, profiles, XI 9; 6c, left mandible in anterior view, XI 85; 6d, head in anterior view, X76. 7. Profiles of mature larvae to show size range (hairs omitted), X8. 7a, Major worker; 7b, minor worker. Figures 6-7. Fifth Instar (Mature Larva). 6a (hairs omitted) and 6b, profiles, XI 9; 6c, left mandible in anterior view, XI 85; 6d, head in anterior view, X76. 7. Profiles of mature larvae to show size range (hairs omitted), X8. 7a, Major worker; 7b, minor worker. like, thorax narrowed abruptly and turned ventrally, T1 about same diameter as head length. Otherwise larvae with different profiles similar. Anus with small lips. Leg, wing and gonopod vestiges pres- ent. Body hairs with bifid or frayed tip. Antennae small, each with 3 sensilla; at midlength of cranium. Head hairs with slightly curved shaft and 2- or 3-branched tip. Mandibles ectatommoid; heavily sclerotized; apical tooth long and narrowed to a sharp point; sub- apical tooth at end of medial blade, with stout base and sharp apex. Maxillary palp paxilliform with 5 (4 apical and 1 lateral) sensilla; galea stout and digitiform with 2 apical sensilla. Vermessor pergandei (Mayr) Figures 1-6. Labium with mi- 307 heeler & Wheeler — Larvae of Veromes 1987] nute spinules in short to long arcuate rows which are arranged in subtransverse rows; opening of sericteries a moderately long trans- verse slit. Material studied: numerous larvae from Death Valley National Monument, California and Boulder City, Nevada. In studying the living larvae the following characters will be most useful in distinguishing instars: First instar. Lacks hairs on body. Head hairs few (about 20). Second Instar. Body hairs very few, confined to T1 only. Head hairs few (about 35). Third Instar. Body hairs very few, some on each thoracic somite. Fourth Instar. Body profile lacks distinct neck. Entire integument covered with spinules and hairs, the hairs with short frayed tips. Fifth Instar (Mature Larva). Body profile pogonomyrmecoid. Length 4. 3-6. 8 mm. Body hairs with short 2- or 3-branched tip. Sponsored by Biodiversity Heritage Library Biodiversity Heritage Library Copyright & Reuse Copyright Status: Public domain. The BHL considers that this work is no longer under copyright protection. Holding Institution Smithsonian Libraries and Archives Sponsored by Biodiversity Heritage Library This file was generated 28 March 2024 at 20:33 UTC Wheeler, George C. and Wheeler, Jeanette. 1987. "Young Larvae of Veromessor Pergandei (Hymenoptera: Formicidae: Myrmicinae)." Psyche 94, 303–307. https://doi.org/10.1155/1987/94737. Summary The larva of each of five instars of Veromessor pergandei is de- scribed in detail and illustrated. In the fifth instar there are two types of body profiles. The instars of living larvae may be differentiated by a few easily observed characters. Copyright & Reuse py g Copyright Status: Public domain. The BHL considers that this work is no longer under copyright protection. This document was created from content at the Biodiversity Heritage Library, the world's largest open access digital library for biodiversity literature and archives. Visit BHL at https://www.biodiversitylibrary.org. This file was generated 28 March 2024 at 20:33 UTC
https://openalex.org/W2768541452	https://europepmc.org/articles/pmc5739683?pdf=render	English	null	RNA-sequencing reveals genome-wide long non-coding RNAs profiling associated with early development of diabetic nephropathy	Oncotarget	2,017	cc-by	9,590	RNA-sequencing reveals genome-wide long non-coding RNAs profiling associated with early development of diabetic nephropathy Copyright: Tang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License 3.0 (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Copyright: Tang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License 3.0 (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. ABSTRACT Background: Diabetic nephropathy (DN) seriously threatens the lives of patients, and the mechanism of DN remains largely unknown because of the complex regulation between long non-coding RNA (lncRNA) and protein-coding genes. In early development of diabetic nephropathy (DN), pathogenesis remains largely unknown. Results: We used RNA-sequencing to profile protein-coding and lncRNA gene transcriptome of mouse kidney proximal tubular cells during early stage of DN at various time points. Over 7000 protein-coding and lncRNA genes were differentially expressed, and most of them were time-specific. Nearly 40% of lncRNA genes overlapped with functional element signals using CHIP-Seq data from ENCODE database. Disease progression was characterized by lncRNA expression patterns, rather than protein-coding genes, indicating that the lncRNA genes are potential biomarkers for DN. For gene ontologies related to kidney, enrichment was observed in protein-coding genes co-expressed with neighboring lncRNA genes. Based on protein-coding and lncRNA gene profiles, clustering analysis reveals dynamic expression patterns for kidney, suggesting that they are highly correlated during disease progression. To evaluate translation of mouse model to human conditions, we experimentally validated orthologous genes in human cells in vitro diabetic model. In mouse model, most gene expression patterns were repeated in human cell lines. Conclusions: These results define dynamic transcriptome and novel functional roles for lncRNAs in diabetic kidney cells; these roles may result in lncRNA-based diagnosis and therapies for DN. INTRODUCTION (PTCs) play critical role in onset and progression of DN. Dimensions and function of proximal tubule increase in response to higher glucose reabsorption, which is caused by increased glomerular filtration of glucose [2], inducing glomerular hyperfiltration through tubuloglomerular feedback [3]. In response to hyperglycemia, PTCs exhibit, early behaviors, such as cell cycle arrest, hypertrophy, and senescence phonotype [4], which are linked to late inflammation, fibrosis, and apoptosis [5]. Kidney injuries Diabetic nephropathy (DN) is major serious complication of diabetes and is the most common cause of end-stage renal disease with poor prognosis and high cost for therapy [1]. However, considering incomplete understanding of DN pathogenesis, early efficacious diagnosis and treatment are still unresolved issues. Emerging evidence show that proximal tubular cells www.impactjournals.com/oncotarget RNA-sequencing reveals genome-wide long non-coding RNAs profiling associated with early development of diabetic nephropathy www.impactjournals.com/oncotarget/ Oncotarget, 2017, Vol. 8, (No. 62), pp: 105832-105847 Research Paper www.impactjournals.com/oncotarget/ Experiments workflow and animal models The workflow was shown in Figure 1-A and the purity of sorted cells was shown in Supplementary Figure 1. All mice in model group developed remarkably high blood glucose (>300 mg/dl) two weeks after first STZ injection (Figure 1-B), indicating successful establishment of mouse diabetes models for subsequent experiments. Urine albumin–to–urine creatinine ratio (ACR) level was also gradient-increased two weeks after STZ injection and peaked at week 8 (Figure 1-C), indicating that kidney injury appeared in early stage of mouse diabetes models. In PAS staining at 2, 4, and 8 weeks of our study, tubular tissue exhibited enlarged lumen, bared cells, and reduced or lost microvilli at different degrees. Statistical difference of TDI with value obtained of 0 w has reached (Figure 1-D), indicating existence of tubular injury in DN development [23]. Tubulointerstitium fibrosis is late- stage character lesion of DN. Masson trichrome–staining showed absence of fibrosis of tubulointerstitium (Figure 1-E). Results indicated that DN was in early stage. Strong correlation between LncRNA and diabetes was reported in recent literature. Morán et al. identified more than 1100 lncRNAs in human islets and several lncRNAs, which were dysregulated in islets from type 2 diabetes patients [16]. Data from study by Xu et al. showed that silencing lncRNA-nc021972 alleviated activation of P2X7 receptor and subsequent tumor necrosis factor-α and interleukin-6 release in in vitro DN models [17]. In recent studies, evidence also demonstrated involvement of lncRNAs in regulation of pathologic genes associated with DN. Surveys by Alvarez et al. showed that in mesangial cells, lncRNA plasmacytoma variant translocation 1 increases plasminogen activator inhibitor 1 and transforming growth factor beta 1, which are two primary contributors to extracellular matrix accumulation in glomeruli under hyperglycemic conditions [18]. Long et al. reported that peroxisome proliferator-activated receptor gamma coactivator alpha (PGC-1α) is functionally regulated by lncRNA taurine-upregulated gene 1 (Tug1). Direct interaction between PGC-1α and Tug1 can modulate mitochondrial bioenergetics in podocytes in DN models [19]. Wang et al. discovered that lncRNA CYP4B1-PS1-001 and ENSMUST00000147869 were significantly downregulated in response to early DN in db/db mice, whereas overexpression of two lncRNAs inhibited proliferation and fibrosis of mesangial cells [20, 21]. www.impactjournals.com/oncotarget Oncotarget 105832 understanding pathogenesis and novel treatment of DN. Next-generation sequencing (NGS) technologies provide edge-cutting method for gene expression research, especially those including lncRNAs under pathophysiological conditions [22]. Thus, aim of this study was threefold. First, to discover genome-wide mRNA and lncRNA profiles of fresh isolated PTCs in animal model during dynamic progression of DN by using NGS. Second, to identify novel lncRNAs, evaluate importance of LncRNA and predict relationship between mRNA and lncRNA during dynamic disease progression by using algorithms. Third, to validate the identified novel RNAs may also play role in pathogenesis of human DN. Our first exploration of genome-wide lncRNAs in DN progression may drive discovery of new early biomarkers and novel therapeutic strategies. are worsened by underlying pathogenic mechanisms for PTC metabolic disorder and abnormal response involving numerous genes and their precise transcriptional regulation networks. However, available information is insufficient to describe changes in genes and regulation during progress of DN. Long non-coding RNAs (lncRNAs) are novel class of functional RNAs; these transcripts measure more than 200 nt and do not code for proteins. lncRNAs and protein- coding transcripts exhibit many similarities, which are as follows: a) presence of 5′ cap and 3′ poly adenosine tail structures; b) transcribed by RNA-polymerase (Pol) II; c) can be spliced at canonical splicing sites [6]. Growing knowledge suggests that lncRNA may play critical role in growth, development, senescence, and disease [7, 8]. And many computational tools has been developed [9-11]. lncRNAs have the following primary functions on gene expression: as regulators of transcription via chromatin modulation [12] and epigenetic modification [13], as regulators of mRNA processing via influence splicing patterns of mRNAs [14], and as modulators of post- transcriptional control [15]. Complex functions of lncRNA are far beyond current understanding and require further characterization. Systematic profiling of mRNAs during early development of DN We performed factorial RNA-Seq study to monitor transcriptome for mouse kidney PTCs during early development of DN. Specifically, we monitored disease progression at four time points after inducing DN: 0, 2, 4, and 8 weeks (denoted by W0/W2/W4/W8). For each time point, two biological replicate RNA-Seq data were generated. Using unique mapped reads to estimate expression levels of mRNA genes, we identified 21,599 mRNA genes, of which 9,625 were expressed at FPKM value≥ 1 in at least one sample. Quantification results are consistent with strong correlation of gene expression between replicate However, above sporadic studies provided insufficient information in genomic changes of lncRNA profiles for specific kidney cells during DN progression. Therefore, genome-wide discovery of lncRNA is needed to identify new concepts and opportunities for profoundly www.impactjournals.com/oncotarget Oncotarget 105833 W8 versus W0 comparison (N=3068, FDR <0.05) followed by W4 (W2) versus W0 comparison (N=1068 and 1516) (Figure 2-A), suggesting that more disturbed transcriptome of mRNAs is associated with progression of kidney disease. Then, we checked overlapping among samples (mean correlation coefficient = 0.98; mean standard deviation = 0.006; Supplementary Figure 2). Taking W0 as control, we identified differentially expressed protein-coding genes (Materials). We observed largest number of differentially expressed genes in Figure 1: Workflow and phenotypes of animal models. (A) Workflow of experiments. a. Construction of animal models; b. examination of phenotype; c. PTC isolation; d. libraries for preparation of RNA-sequencing; e. next-generation sequencing; f. bio-information analysis. (B) Fasting blood sugar over time. Data are represented as mean ±SEM. p < 0.05 by t-test versus control at 0 week. #p < 0.05 by t-test versus control at the same week. n = 6 for each group. (C) ACR over time. Data are represented as mean ±SEM. p < 0.05 by t-test versus control at 0 week. #p < 0.05 by t-test versus control at the same week. n = 6 for each group. (D) Left: PAS-stained sections in each group. Images display kidney sections of each group at 200× magnification. White arrow indicates normal tubule, and black arrow indicates injured tubule. Views with high magnification are shown at lower left. Time course are labeled as a, b, c, and d; letters correspond to 0, 2, 4, and 8 weeks. Right: Tubule injuries were evaluated for widened lumen, atrophy, or thickened basement membranes. Tubular injuries are indicated with arrows. Bar graph shows TDI for each group. Systematic profiling of mRNAs during early development of DN www.impactjournals.com/oncotarget Oncotarget 105834 differentially expressed genes at different time points (Figure 2-B). W2 and W4 have time-specific genes of 186 and 94, respectively. By contrast, W8 has 1761 specific differentially expressed genes. This result suggests very modest disturbance to transcriptome between W2 and W4. expressed lncRNA genes (Figure 3-D). Compared with protein- coding genes, lncRNA genes presented similar functions in gene ontology. However, comparison indicated that programmed cell death was at W2 for lncRNA genes, whereas function was enriched at W8 for protein-coding genes. Results suggested that dysfunction of pathways for kidney is initialized by disturbing regulators, such as lncRNA genes, beforehand. To further characterize differentially expressed genes obtained from RNA-Seq analysis, we manually curated gene list associated with kidney by literature research, where 262 protein-coding genes were included (Supplementary Table 3). And, 76 of them are differentially expressed based on our analysis (Figure 2-C). For example, Angpt1, Stat1, Agtr1a, Cdc42, Nox4, and Shc1 are differentially expressed in at least one time point (Figure 2-E). Nox4 was reported to be upgraded in several kinds of DN models and played key factor in reactive oxygen species-related signal pathway [24-26]. To assess expression of orthologous lncRNA genes in humans, we used BLAST Software [27] to obtain orthologous lncRNA genes between mice and humans. We validated the expression of orthologous lncRNA genes in human cell line using qPCR (Figure 3-E). We validated the expression of orthologous lncRNA genes in human cell line. glucose stimulation and had similar expression pattern with orthologous genes of mouse. Considering these results, we concluded that lncRNA data from mouse diabetic model was also partly applicable to human DN. To assess functions of differentially expressed genes, we performed gene ontology analysis for time-specific protein-coding genes (Figure 2-D, Materials). W4-specific gene functions are involved in phosphorylation, peptidyl- serine modification, and enzyme-linked receptor protein signaling pathway, whereas those for W8-specific genes are transmembrane transport, programmed cell death, oxidation reduction, and cell cycle. Orthologous lncRNA genes in humans underwent motif searching (Figure 3-F). Motifs searching were calculated using TOMTOM software [28]. Surprisingly, some lncRNA has several possible motif sites for specific transcriptional factor binding. For instance, lncRNA NONHSAG053901 has four possible sites in its transcriptome for transcription factor (TF) early growth response protein 1 (EGR1)-binding. EGR1 was found to be responsible for activation of heparanase promoter under diabetic conditions [29]. Differentially expressed lncRNAs are enriched by functional signals of ChIP-Seq lncRNA genes are acknowledged as key regulators for diseases [30, 31]. To further characterize lncRNA signatures, we analyzed TSS of differentially expressed lncRNA genes for presence of histone modifications. Actively transcribed lncRNA genes were associated with promoter-associated histone modification (trimethylation of histone 3 at Lys4, H3K4me3) or enhancer-associated histone modification (monomethylation of histone 3 at Lys4, H3K4me1). Therefore, we used publicly available H3K4me1 and H3K4me3 ChIP-Seq data for kidney information from ENCODE database. Systematic profiling of mRNAs during early development of DN Data are represented as mean ±SEM. p < 0.05 by t-test versus control in 0 week. n = 6 for each group. (E) Left: Masson-trichrome-stained kidney sections in each group. Images display kidney sections of each group at 200× magnification. White arrow indicates normal tubule, and black arrow indicates injured tubule. Views with high magnification are shown at lower left. Time courses are labeled as a, b, c, and d, corresponding to 0, 2, 4, and 8 weeks. Right: Bar graph shows relative interstitial volume for each group. Data are represented as mean ±SEM. n = 6 for each group. Figure 1: Workflow and phenotypes of animal models. (A) Workflow of experiments. a. Construction of animal models; b. examination of phenotype; c. PTC isolation; d. libraries for preparation of RNA-sequencing; e. next-generation sequencing; f. bio-information analysis. (B) Fasting blood sugar over time. Data are represented as mean ±SEM. p < 0.05 by t-test versus control at 0 week. #p < 0.05 by t-test versus control at the same week. n = 6 for each group. (C) ACR over time. Data are represented as mean ±SEM. p < 0.05 by t-test versus control at 0 week. #p < 0.05 by t-test versus control at the same week. n = 6 for each group. (D) Left: PAS-stained sections in each group. Images display kidney sections of each group at 200× magnification. White arrow indicates normal tubule, and black arrow indicates injured tubule. Views with high magnification are shown at lower left. Time course are labeled as a, b, c, and d; letters correspond to 0, 2, 4, and 8 weeks. Right: Tubule injuries were evaluated for widened lumen, atrophy, or thickened basement membranes. Tubular injuries are indicated with arrows. Bar graph shows TDI for each group. Data are represented as mean ±SEM. p < 0.05 by t-test versus control in 0 week. n = 6 for each group. (E) Left: Masson-trichrome-stained kidney sections in each group. Images display kidney sections of each group at 200× magnification. White arrow indicates normal tubule, and black arrow indicates injured tubule. Views with high magnification are shown at lower left. Time courses are labeled as a, b, c, and d, corresponding to 0, 2, 4, and 8 weeks. Right: Bar graph shows relative interstitial volume for each group. Data are represented as mean ±SEM. n = 6 for each group. Systematic profiling of mRNAs during early development of DN Results implied that lncRNAs possibly directly interact with transcriptional factor and play important role in regulation of gene transcription or cell signal pathway. To assess application of the mouse DN model, we validated differentially expressed genes by using human cell line in vitro diabetic model. We selected six differentially expressed genes with mono decreasing (increasing) gene expression and obtained their orthologous genes in human from Mouse Genome Informatics database. Figure 2-F shows expression levels of orthologous genes in vitro diabetic model by using qPCR. Surprisingly, all genes were differentially expressed after high-glucose stimulation and presented similar tendency (up- or downregulation) as that in mouse RNA-seq. These results indicated that mRNA data from RNA-seq in mouse DN model may provide similar evidence for human DN. www.impactjournals.com/oncotarget Systematic profiling of lncRNAs during early development of DN We identified 29,273 lncRNA genes, of which 15,138 were expressed at FPKM value of ≥ 0.5 in at least one sample. Hierarchical clustering revealed that transcriptome profiles of kidneys were well separated during disease progression, and samples were clustered into three groups: W0, W2 and W4, and W8 (Figure 3-A). Figure 3-B indicated the largest number of differentially expressed lncRNA genes in W8 versus W0 comparison (N=3426, FDR<0.05) followed by W4 (W2) (N=875, 1199, FDR<0.05) (Figure 3-B). Figure 3-C shows overlapping of differentially expressed lncRNA genes, and the pattern is consistent with that for mRNA genes. Over one third of TSSs of differentially expressed lncRNA genes showed overlapping with significant peaks (39.7%, Methods and Materials) for at least one type of histone modifications, a proportion similar to that of previously annotated lncRNA genes associated with such histone modifications (37.5% [32], 37% [31]). H3K4me1 modifications showed distribution of broader density around TSSs than that of H3K4me3 modifications (Figure 4-A). Figure 4-B summarizes density functions of corresponding histone modifications. These results Neighbored protein-coding genes were used to analyze gene-ontology of time-specific differentially www.impactjournals.com/oncotarget Oncotarget 105835 Figure 2: Transcriptome profiles of mRNAs associated with disease progression. (A) Number of differentially expressed mRNA genes at various time points compared with control (W0). P-value cutoff is 0.05. (B) Venn diagram of differentially expressed mRNA genes at different times. (C) Overlapping between differentially expressed mRNA genes and reported mRNA genes associated with kidney disease progression. (D) Gene ontology enrichment for week-specific and common differentially expressed mRNA genes, presented as -log10 hypergeometric P-value for enrichment. (E) Gene expression of some key genes that are highly related to kidney. y-axis denotes FPKM value, x-axis is for four time points, and error bar is standard deviation, * indicates p-value < 0.05 by comparison with W0. (F) Validation for differentially expressed mRNAs in human PTCs (HK2)Left portion: Results from RNA-seq in mouse DN models (p<0.05 versus W0). Right portion: Results from qPCR in vitro diabetic models. NG, cultured with normal glucose; HG, cultured with 30 mM glucose for 48 h.p<0.05 versus NG, n=3. Figure 2: Transcriptome profiles of mRNAs associated with disease progression. (A) Number of differentially expressed mRNA genes at various time points compared with control (W0). P-value cutoff is 0.05. (B) Venn diagram of differentially expressed mRNA genes at different times. Systematic profiling of lncRNAs during early development of DN (C) Overlapping between differentially expressed mRNA genes and reported mRNA genes associated with kidney disease progression. (D) Gene ontology enrichment for week-specific and common differentially expressed mRNA genes, presented as -log10 hypergeometric P-value for enrichment. (E) Gene expression of some key genes that are highly related to kidney. y-axis denotes FPKM value, x-axis is for four time points, and error bar is standard deviation, * indicates p-value < 0.05 by comparison with W0. (F) Validation for differentially expressed mRNAs in human PTCs (HK2)Left portion: Results from RNA-seq in mouse DN models (p<0.05 versus W0). Right portion: Results from qPCR in vitro diabetic models. NG, cultured with normal glucose; HG, cultured with 30 mM glucose for 48 h.p<0.05 versus NG, n=3. Expression signature of lncRNAs, but not mRNAs, characterizes DN progression demonstrated that differentially expressed lncRNA genes enriched functional DNA elements. We then determined difference among differentially expressed genes, non-differentially expressed genes, non- expressed genes, random transcripts for both protein- coding genes, and lncRNA genes for each time point; results are shown in Figure 4-C. In case of protein-coding genes, differentially expressed genes were associated with broader distributions of density of histone modification than non-differentially expressed genes at all time points. In case of lncRNA genes, similar tendency was observed at all time points. Comparison between protein-coding genes and lncRNAs indicated that differentially expressed lncRNAs had highest level of H3K4me3 modification among all lncRNA genes, where same observation was not observed in differentially expressed protein-coding genes (Figure 4-C). Figure 3A suggests that lncRNA genes may serve as biomarkers in discriminating disease progression. Therefore, we clustered samples on the basis of both protein-coding genes and lncRNA genes (Figure 5-A). Branch containing samples of W0 and W2 were also well segregated, whereas W4 and W8 samples cannot be divided correctly. We next constructed samples on the basis of differentially expressed protein-coding and lncRNA genes (Figure 5-B). Samples at all time points were correctly segregated when we cut the dendrogram at dashed line. Although samples were correctly classified, W2 and W4 samples were separated; this observation cannot be explained because disease progressed from W2 to W4. To determine inaccuracy, we next constructed samples based on protein-coding genes that were differentially expressed in at least one time point (Figure In summary, our results indicate that transcriptional landscapes of mouse kidney were characterized by lncRNA genes. Figure 4: TSSs of differentially expressed lncRNA genes are enriched by active chromatin signal. (A) ChiP-Seq analysis of histone- modification profiles (H3K4me1 and H3K4me3) at TSSs of lncRNA genes, which are differentially expressed in at least one time point. (B) Density functions for lncRNA genes of H3K4me1 and H3K4me3. (C) ChIP-Seq “metaplots” of histone-mark density at TSSs of protein- coding and lncRNA genes, which are stratified by gene expressions: differentially expressed genes, non-expressed genes, week-specific differentially expressed genes, expressed but not differentially expressed genes, and random genes. Figure 4: TSSs of differentially expressed lncRNA genes are enriched by active chromatin signal. (A) ChiP-Seq analysis of histone- modification profiles (H3K4me1 and H3K4me3) at TSSs of lncRNA genes, which are differentially expressed in at least one time point. (B) Density functions for lncRNA genes of H3K4me1 and H3K4me3. www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget Oncotarget 105836 gure 3: Transcriptome profiles of lncRNA genes associated with disease progression. (A) Hierarchical clustering of lncRNA genes at are differentially expressed in at least one time point by comparison with that at W0; rows and columns correspond to samples and cRNA genes, respectively. (B) Number of differentially expressed lncRNA genes at various time points compared with control (W0) th corrected P-value cutoff of 0.05. (C) Venn diagram of differentially expressed lncRNA genes at different times. (D) Gene ontology richment for week-specific and common differentially expressed lncRNA genes, presented as −log10 hypergeometric P-value for richment. (E) Validation for differentially expressed lncRNAs in PTCs (HK2)Left portion: Results from RNA-seq in mouse DN models p<0.05 versus W0). Right portion: Results from qPCR in vitro diabetic models. NG, cultured with normal glucose; HG, cultured with 30 M glucose for 48 h. p<0.05 versus NG, n=3. (F) Motifs searched for validated lncRNAs in PTCs (HK2). TF, transcriptional factor. Sites, mber of possible motifs in one LncRNA sequence. Figure 3: Transcriptome profiles of lncRNA genes associated with disease progression. (A) Hierarchical clustering of lncRNA genes that are differentially expressed in at least one time point by comparison with that at W0; rows and columns correspond to samples and lncRNA genes, respectively. (B) Number of differentially expressed lncRNA genes at various time points compared with control (W0) with corrected P-value cutoff of 0.05. (C) Venn diagram of differentially expressed lncRNA genes at different times. (D) Gene ontology enrichment for week-specific and common differentially expressed lncRNA genes, presented as −log10 hypergeometric P-value for enrichment. (E) Validation for differentially expressed lncRNAs in PTCs (HK2)Left portion: Results from RNA-seq in mouse DN models (p<0.05 versus W0). Right portion: Results from qPCR in vitro diabetic models. NG, cultured with normal glucose; HG, cultured with 30 mM glucose for 48 h. p<0.05 versus NG, n=3. (F) Motifs searched for validated lncRNAs in PTCs (HK2). TF, transcriptional factor. Sites, number of possible motifs in one LncRNA sequence. www.impactjournals.com/oncotarget Oncotarget 105837 Expression signature of lncRNAs, but not mRNAs, characterizes DN progression (C) ChIP-Seq “metaplots” of histone-mark density at TSSs of protein- coding and lncRNA genes, which are stratified by gene expressions: differentially expressed genes, non-expressed genes, week-specific differentially expressed genes, expressed but not differentially expressed genes, and random genes. www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget Oncotarget 105838 5-C). W0 and W8 samples were correctly classified, whereas W2 and W4 samples were mixed. Results indicated that protein-coding genes were inadequate for characterizing disease progression. We hypothesized that lncRNA genes can be used to characterize disease progression. To investigate inaccuracy factor, we next constructed samples based on the differentially expressed lncRNA genes (Figure 5-D). All samples were correctly classified when dendrogam was cut at the dashed line. W2 and W4 samples were also successfully segregated. separated by distance of > 1M base or located on various chromosomes). Expression of lncRNA genes was more closely correlated than protein-coding genes in trans. In all cases, bias toward correlations was significantly higher than that obtained for control set of trans correlations, in which expression of lncRNA and protein-coding genes was randomly shuffled (Figure 6-A). We then analyzed cis correlations of expression (gene pair located within genomic window of 100K bases). We observed higher proportion of correlations among cis correlations than among trans correlation for both lncRNA-gene–protein- coding gene pairs and protein-coding gene–protein- coding gene pairs (Figure 6-A). These results indicate that lncRNA genes possibly perform functions via combination of multiple lncRNA genes. Overall, these data demonstrated that lncRNA expression can be used to define developmental relationships, while protein-coding genes are not. Correlation of lncRNA and protein-coding gene expression Gene ontology enrichment analysis focused on protein-coding genes with strong cis correlations with protein-coding genes, and results revealed that enrichment for gene-encoding products involved oxidation and linoleic acid metabolic process. By contrast, genes with cis correlations with lncRNA genes were significantly To investigate co-expression patterns of lncRNA and protein-coding genes, we computed pairwise expression correlations across all RNA-Seq samples. We first analyzed trans correlations of expression (genes Figure 5: Disease progression is characterized by global expression patterns of lncRNA genes rather than mRNA genes. Sample clustering analysis based on gene expression of various combination of protein-coding and lncRNA genes, where dashed line is cut line for dendrogram, and samples surrounded by dashed circles are misclassified. (A) Protein-coding and lncRNA genes. (B) Differentially expressed protein-coding and lncRNA genes in at least one time point. (C) Differentially expressed protein-coding genes. (D) Differentially expressed lncRNA genes. Figure 5: Disease progression is characterized by global expression patterns of lncRNA genes rather than mRNA genes. Sample clustering analysis based on gene expression of various combination of protein-coding and lncRNA genes, where dashed line is cut line for dendrogram, and samples surrounded by dashed circles are misclassified. (A) Protein-coding and lncRNA genes. (B) Differentially expressed protein-coding and lncRNA genes in at least one time point. (C) Differentially expressed protein-coding genes. (D) Differentially expressed lncRNA genes. DISCUSSION Discovering new biomarkers and therapy targets is crucial for diagnosis and treatment of DN. Progress, however, is slow. Recently, research on lncRNAs illuminated understanding of DN. Few lncRNAs have close relationship with DN progression; these lncRNAs include lncRNA PVT1 [18], lnc-MGC [35], and Tug1 [19], etc.. Difficulty arises from exposing complete structure of lncRNAs in DN process these dispersive studies, and our understanding is presently in its infancy. Thus, large-scale data on lncRNAs and mRNAs in DN need to be gathered for better understanding of this disease. Recently, Ding et al [36] investigate lncRNA expression in DN using the microarray data. However, they use the mixed cells from kidney, rather than the PTCs. Hence, we revealed genome- wide profiles of lncRNAs and mRNAs from isolated fresh PTCs using NGS in DN process for the first time. Transcriptome information from fresh isolated tissues can represent actual appearance of disease to maximum extent [37]. Therefore, this study is novel and pioneering on global views about lncRNAs and mRNAs in DN research. To assess correlation of lncRNA and protein-coding genes, we validated gene expression of orthologous genes in humans using qPCR (Figure 6-D). We showed that three typical pairs of lncRNAs and protein-coding genes were consistent with those trends in mice. To our knowledge, this report represents first investigation of protein-coding and lncRNA elements in mouse kidney PTCs during early development of DN. And, we discovered more than 3,502 differentially expressed protein-coding genes, in which 76 genes are reported in previous studies. We also identified 3,818 differentially expressed lncRNA genes. Interestingly, 390 (556) lncRNA genes exhibited mono-decreasing (mono- increasing) expression levels. Patterns indicate dynamics of genes during disease progression, suggesting important role of lncRNAs during initial stages of diabetes. Functions of lncRNA genes include cell proliferation and cell death, and they are closely associated with diabetes. www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget Oncotarget 105839 associated with these functional annotations, such as response to cytokine stimulus, protein kinase cascade, and transmembrane transport (Figure 6-B). 7-C provides two schematic examples of co-expression module consisting of protein-coding and lncRNA genes, and functions of lncRNAs can be inferred from protein- coding genes within modules based on topology. Overall, these results provide information for identification of lncRNA candidates and formulation of hypotheses for functional studies to elucidate role of lncRNAs in DN. p ( g ) To further investigate co-expression signature of genes, we delineated expression profiles of protein- coding genes and differentially expressed lncRNA genes in at least one time point (FDR<0.05). We used model- based gene clustering to obtain patterns for expression profiles [33], which are presented in Figure 6-C. In total, we discovered 21 modules, which were classified into six groups. Four specific profiles showed significant enrichment for lncRNA genes (p-value<0.05, proportion test) compared with proportion of lncRNA genes among all differentially expressed genes. These profiles were upregulated at W8 (profile 9, 10, 13, and 14), suggesting that more lncRNA genes became dysfunctional as disease progressed. Differentially expressed lncRNA genes showed significant enrichment (P < 0.05; relative to all lncRNA genes) for loci neighboring protein-coding genes, whose products were involved in transmembrane transport (p-value=9.7E-5, hypergeometric test), programmed cell death (p-value=8.4E-3), nitrogen compound biosynthetic process (p-value=5.6E-4), and cell cycle (p-value=1.0E-7). In summary, global co-expression analysis and gene- expression profiling suggested notable and previously unappreciated role for lncRNAs in early development of DN. Co-expression network analysis reveals modules of lncRNA and protein-coding genes Correlation-based approaches are widely used to infer function of lncRNAs. Thus, we used network analysis by using WGCNA algorithm [34], which identified modules of genes with strong co-expression. Eigengene measures expression profile of co-expression module, and such profiles can be used to rank individual genes within modules. Screening for genes with high module membership is useful strategy for identification of genes of interest. Prior information was used to annotate lncRNA genes. Recently, Orom et al. [38] used GENCODE annotation of human genome to characterize over a thousand lncRNAs, and discovered that significant portion of lncRNAs have cis-regulatory enhancer properties. To characterize the differentially expressed lncRNA genes, we analyzed TSSs of lncRNAs for presence of histone modifications. By using ChiP-Seq data (H3K4me1, H3K4me3) from ENCODE database for kidney [39]. And, we discovered that nearly 40% of differentially expressed lncRNA genes overlap with histone modification signals. Result demonstrates that obtained lncRNA genes are key regulators for kidney disease. Moreover, we showed Of 102 modules discovered by WGCNA, four modules were selected for further analysis. Figure 7-A shows heatmap of protein-coding and lncRNA co-expression network. As shown in Figure 7-B, in eigengenes of four selected co-expressed modules, expression level of modules mono-decreased (increasing), indicating disease progression. These modules contained protein-coding genes associated with cell functions, including transmembrane transport (p-value=0.01), lipid biosynthetic process (p-value=7.2E-4), oxidation reduction (p-value=0.01), and cell division (p-value=8.7E-3). Figure www.impactjournals.com/oncotarget Oncotarget 105840 that lncRNA genes are more accurate than mRNA genes in discriminating disease progression. These results are consistent with previous study [31], where lncRNAs discriminate cell development. Functions of lncRNA genes are cell proliferation, cell death, oxidation reduction, and phosphorylation, and these are closely associated with g g n discriminating disease progression. These results are consistent with previous study [31], where lncRNAs p genes are cell proliferation, cell death, oxidation reductio and phosphorylation, and these are closely associated wi Figure 6: lncRNA genes are co-expressed with protein-coding genes during disease progression. (A) Boxplot of pairwi Spearman expression correlations between genes of different classes in trans (left) and cis (right): lncRNA–to–lncRNA, lncRNA–mRN mRNA–mRNA, and random mRNA–lncRNA. (B) Gene ontology enrichment for protein-coding genes in trans and correlated with lncRNA first column, mRNA–lncRNA), and nearest protein-coding genes in trans and correlated with lncRNA genes (lncRNA–lncRNA) present as −log10 hypergeometric P value for enrichment. (C) Model-based expression profiles (Profiles 1–22; Groups I–VI) of protein-codin and lncRNA genes expressed differentially during disease progression. Co-expression network analysis reveals modules of lncRNA and protein-coding genes Numbers above plots indicate profile number plus total genes an ncRNA genes in profile (in parentheses: total lncRNA). y-axis is Z-score of gene expression, and x-axis is time point. In each plot, gr ine is expression level of gene in module, and red line is average expression level of genes within module. Red asterisks indicate profil howing enrichment for lncRNA genes (P < 0.05 versus proportion of lncRNAs among all differentially expressed genes). (D) Validati or predicted mRNA–lncRNA pairs in PTCs (HK2) Mouse: Predicted mRNA–lncRNA pairs from RNA-seq in mouse DN models. Le portion: mRNA results (all differential expressions are significant). Middle portion: lncRNA results. Right portion: Correlation coefficie between mRNA and lncRNA. All P values < 0.01. Human: Validation for predicted mRNA–lncRNA pairs in PTCs (HK2) down-regulat ncRNAs by qPCR. NG, cultured with normal glucose; HG, cultured with 30 mM glucose for 48 h. p<0.05 versus NG, n=3. Figure 6: lncRNA genes are co-expressed with protein-coding genes during disease progression. (A) Boxplot of pairwise Spearman expression correlations between genes of different classes in trans (left) and cis (right): lncRNA–to–lncRNA, lncRNA–mRNA, mRNA–mRNA, and random mRNA–lncRNA. (B) Gene ontology enrichment for protein-coding genes in trans and correlated with lncRNAs (first column, mRNA–lncRNA), and nearest protein-coding genes in trans and correlated with lncRNA genes (lncRNA–lncRNA) presented as −log10 hypergeometric P value for enrichment. (C) Model-based expression profiles (Profiles 1–22; Groups I–VI) of protein-coding and lncRNA genes expressed differentially during disease progression. Numbers above plots indicate profile number plus total genes and lncRNA genes in profile (in parentheses: total lncRNA). y-axis is Z-score of gene expression, and x-axis is time point. In each plot, gray line is expression level of gene in module, and red line is average expression level of genes within module. Red asterisks indicate profiles showing enrichment for lncRNA genes (P < 0.05 versus proportion of lncRNAs among all differentially expressed genes). (D) Validation for predicted mRNA–lncRNA pairs in PTCs (HK2) Mouse: Predicted mRNA–lncRNA pairs from RNA-seq in mouse DN models. Left portion: mRNA results (all differential expressions are significant). Middle portion: lncRNA results. Right portion: Correlation coefficient between mRNA and lncRNA. All P values < 0.01. Human: Validation for predicted mRNA–lncRNA pairs in PTCs (HK2) down-regulated lncRNAs by qPCR. NG, cultured with normal glucose; HG, cultured with 30 mM glucose for 48 h. *p<0.05 versus NG, n=3. www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget Oncotarget 105841 DN. www.impactjournals.com/oncotarget Co-expression network analysis reveals modules of lncRNA and protein-coding genes In addition, current study indicates that lncRNA genes exhibited earlier symptoms of disease compared with that of mRNA genes, which may provide novel evidence for early diagnosis and therapy of DN. differential expression pattern as their orthologous lncRNA in mouse. Interestingly, the lncRNAs have not been reported in DN before. Poor conservation of lncRNAs across different species challenges application of animal sequencing data to human disease [6, 40]. However, in clinical practice, in patients with early DN, conditions prevent obtaining sufficient kidney samples To further investigate application values of mouse model, we validated several that human orthologous lncRNAs in vitro diabetes model. And, they had similar Oncotarget 105842 www.impactjournals.com/oncotarget Figure 7: Network analysis identifies co-expression modules of lncRNA and protein-coding genes. (A) Heatmap of WGCNA topological overlap matrix, where each row (column) corresponds to a gene, and bar color indicates genes clustered as module. (B) Eigengene of selected modules represents gene expression level of genes within a module. (C-D) Screening strategies for identification of candidate lncRNA genes and lncRNA-gene–protein-coding gene co-expression associations in specific modules for further functional studies presented as Circos plots from analysis of HSPC module (broken lines in circumference indicate individual chromosomes). Figure 7: Network analysis identifies co-expression modules of lncRNA and protein-coding genes. (A) Heatmap of WGCNA topological overlap matrix, where each row (column) corresponds to a gene, and bar color indicates genes clustered as module. (B) Eigengene of selected modules represents gene expression level of genes within a module (C D) Screening strategies for identification Figure 7: Network analysis identifies co-expression modules of lncRNA and protein-coding genes. (A) Heatmap of WGCNA topological overlap matrix, where each row (column) corresponds to a gene, and bar color indicates genes clustered as module. (B) Eigengene of selected modules represents gene expression level of genes within a module. (C-D) Screening strategies for identification of candidate lncRNA genes and lncRNA-gene–protein-coding gene co-expression associations in specific modules for further functional studies presented as Circos plots from analysis of HSPC module (broken lines in circumference indicate individual chromosomes). www.impactjournals.com/oncotarget Oncotarget 105842 assay kit (Albuwell M, Exocell) and creatinine companion kit (Exocell), respectively, per manufacturer’s instructions [51]. for deep sequencing. Our study demonstrates that validation of orthologous lncRNAs from animal data is still an efficacious way to discover novel biomarkers in human DN. lncRNAs exert their effect by direct interaction with specific TFs [41, 42]. Co-expression network analysis reveals modules of lncRNA and protein-coding genes In recent report, hypoxia-regulated lncRNA linc-p21 was shown to physically interact with HIF-1-alpha TFs to control metabolism in tumor cells [43]. We discovered several enriched important TF motifs for lncRNAs. These TFs were previously reported as key regulation factors that closely related to cell proliferation, apoptosis, inflammation, fibrosis, and other signaling pathways in DN [29, 44-46]. In our study, some of predicted potential lncRNAs–mRNA pairs have also been validated in in human cells in vitro diabetic model. Results imply that orthologous lncRNAs may play similar roles in DN even across different species. Histology Kidney tissues of mice were first placed in 4% paraformaldehyde and then embedded in paraffin. Paraffin-embedded tissues were then sectioned at 3 μm to 5 μm, stained with hematoxylin-eosin, periodic acid–Schiff (PAS), and Masson’s trichrome, successively. Digital images were obtained using Leica DM5000B Microscope System. For each mouse, 10 randomly selected cortical areas were evaluated under 200× magnification. Relative interstitial volume was evaluated by morphometric analysis, and tubules were evaluated as tubules damage index (TDI) to estimate percentage of damaged tubules in blinded manner as previously described [23]. Animals The cortical region of mice kidneys was minced into small pieces. Cortical pieces were softly grinded on 250 μm steel sieve, washed with 1×PBS, and spun down. Precipitation was resuspended in solution of 0.5 mg/ml collagenase I, 0.5 mg/ml dispase II, and 0.1% trypsin (Worthington Bio Co., Lakewood, NJ) in complete RPMI- 1640 medium (Gibco) and incubated at 37 °C for 15 min. After digestion, mixture was filtered through 100 and 40 μm cell strainer (BD Biosciences) sequentially. Single cell suspension was treated with ACK lysing buffer (Lonza), then resuspended in 1×PBS to 2×107 cells/ml. Suspension was first incubated with anti-CD16/32 antibody (3 μl/1× 108 cells) (Biolegend) at 4 °C for 10 min. Then cells were washed and resuspended. Kidney cells were incubated with PE-conjugated CD13 antibody (BD Biosciences), which is specific against aminopeptidase N on PTC microvilli (20 μg/ml) at 4 °C in dark for 30 min. A647 conjugated nephrin antibody (Bioss) was added to prevent podocyte contamination. After labeling, cells were washed and resuspended in 1×PBS supplemented with 2% bovine serum albumin to 1×107 cells/ml. Fluorescent-labeled PTCs were isolated by BD FACSAria™ II flow cytometry cell sorting system (BD Biosciences). [52]. Eight weeks old male DBA/2J mice were purchased from Jackson Laboratory (Bar Harbor, ME) and were housed in barrier facility under National Institutes of Health guidelines. The mice were divided randomly into four groups of 48: 0 week group(Control, twelve mice); 2 week group(Diabetes, twelve mice); 4 week group(Diabetes, twelve mice) and 8 week group(Diabetes, twelve mice). In each group, mixed PTCs from every six mice were used for producing one RNA-sequencing dataset. Thus, each group produced two independent RNA-sequencing datasets. Diabetes was induced with intraperitoneal injections of streptomycin (STZ) based on protocol of Animal Models of Diabetic Complications Consortium [47]. In brief, STZ (Sigma) was dissolved in 0.05 M citrate monosodium (ACROS) buffer immediately before injection. Mice received injections of 50 mg/kg/day STZ for five consecutive days after fasting for 4 h. Low doses of STZ has been previously demonstrated to induce durable hyperglycemia without obvious kidney damage [48, 49]. All mice were fed the same diet and water. Insulin was not given to any of the animals. All animal procedures were approved by the Institutional Animal Care and Use Committee of Sichuan University. www.impactjournals.com/oncotarget Data analysis The protein-coding and lncRNA genes differentially expressed at least on one condition were selected for co-expression network analysis (protein-coding: 3884; lncRNA: 3997). Weighted gene co-expression network analysis algorithm (WGCNA) was adopted with default parameters [34]. Chip-sequence data analysis We downloaded Chip-Seq datasets (Encyclopedia of DNA Elements at UCSC [39]) of H3K4me1 and H3K4me3 histone modifications in kidney from eight- week old adult mouse. We used peak information from bigWig files. For various genes, significant peaks and transcription start sites (TSS) overlapped under conditions of strict overlap (TSS was contained in peak region as defined by MACS [59]). Data for read density heatmaps were obtained for window sizes ±3 kb. Blood glucose and urinary albumin/creatine ratio measurements Sorted PTCs were lysed with Trizol-LS immediately (Ambion), and total RNA was prepared using RNeasy Plus Mini Kit (Qiagen) per manufacturers’ instructions. Total RNA concentration was measured by Qubit 2.0 Fluorometer (Invitrogen), and RNA quality was evaluated using Agilent 2100 Bioanalyzer (Agilent). RNA-seq libraries were prepared with Ovation RNA-Seq System V2 Kit (NuGEN) per manufacturer’s instruction. RNA- seq libraries were analyzed by Bioanalyzer (Agilent) and quantified by quantitative PCR (qPCR) (KAPA). Mice blood glucose levels were measured per week after initial STZ injection using ReliOn Ultima glucose reader (Walmart) for 10 weeks. At each time point before sacrifices, spot urine collections were obtained from mice as previously described [50]. Urinary concentrations of albumin and creatine were determined using mouse- specific microalbuminuria enzyme-linked immunosorbent www.impactjournals.com/oncotarget Oncotarget 105843 High-throughput sequencing was performed using Illumina HiSeq 2500 Genome Sequencers [53]. The data is available at GEO database with accession number GSE95367. expression. For calling differentially expressed gene, we used false discovery rate (FDR) cutoff of 0.05, and minimum FPKM is greater or equal to 1 in at least one sample. Cell culture To evaluate functional relevance of genes, we performed gene ontology enrichment analysis on groups of genes using hyper-geometric test. p-values were corrected through BH test [58] with cutoff value of 0.05. Human kidney epithelial cells (HK2) of proximal tubular origin were purchased from American Type Culture Collection (Manassas) and cultured in RPMI 1640 medium (Gibco) supplemented with 10% fetal bovine serum (Gibco) at 37 °C under 5% CO2. Cells were cultured in complete medium with different concentrations of glucose for further validation experiments at 80% confluence after synchronization. Transcriptome assembly and expression level estimate from sequencing reads Paired-end reads were mapped to mouse genome (mm9) using Tophat [54]. Downstream analyses only included uniquely mapped reads with fewer than two mismatches. Transcripts were assembled using Cufflinks [55] and Refseq (mm9) as source of annotated transcripts. Normalized transcript abundance was calculated using Cufflinks. Fragments Per Kilobase of transcripts per Million mapped reads (FPKM) was used to quantify expression (The procedure for the RNA- sequencing analysis is depicted in Supplementary Figure 3). Gene-level FPKM values were computed by summing up FPKM values of their corresponding transcripts. For lncRNA gene expression, same procedure was repeated by using annotation file of NONCODE 4.0 [56] (FPKM values for mRNA are presented in Supplementary 3, and FPKM values for lncRNA are shown in Supplementary 4). Pair-wise co-expression matrix (Topological Overlap Matrix) was generated and clustered to retrieve modules of highly correlated genes (The dendrogram is illustrated in Supplementary Figure 4). Each module obtained by WGCNA was characterized by expression of representative gene (eigengene, Figure 7-B). For each gene, WGCNA also provides summary statistics and module membership (quantifying how strongly expression profile of gene correlates with that of module) information, which can be used to screen for biologically interesting candidate genes for functional studies. Expression correlation analysis We calculated correlations for mRNA–mRNA, mRNA–lncRNA, and lncRNA–lncRNA pairs. For each pair of genes with non-null expression, non-parametric Spearman correlation was computed using FPKM expression. Both trans (pairs consisting of genes located at distance of > 1 Mb from each other or in different chromosomes) and cis (pairs consisting of genes located within genomic window of 1Mb) correlations were computed. Controls used were correlations of lncRNAs and mRNAs with randomly shuffled expression vectors. Real-time qPCR for mRNA and lncRNA expression Total RNA was purified from HK-2 cells using RNeasy Plus Mini Kit (Qiagen) as described ahead. cDNA was obtained by reverse transcription using iScript cDNA Synthesis Kit (Bio-Rad). Then, cDNA was amplified with specific primers (shown in Supplementary Tables 1&2) and detected using SYBR Green Supermix (Bio-Rad) with BIO-RAD CFX-96 Real Time PCR System (Bio- Rad) under the following conditions: 95 °C for 3 min followed by 40 cycles of 95 °C for 10 s and 52 °C for 30 s. Amount of relative expression of cDNA was calculated using 2−△△Ct method. Expression levels were normalized to glyceralde-hyde-3-phosphate dehydrogenase mRNA as internal control. Ethics approval and consent to participate All research involving animals were conducted according to the Regulations for the Administration of Affairs Concerning Experimental Animals (Ministry of Science and Technology, China, revised in June 2004) and approved by the Institutional Animal Care and Use Committee of Animal Science and Technology, Sichuan University. ACKNOWLEDGMENTS We sincerely appreciate Dr. Kai Tan, Peng Gao, and Changya Chen from the university of Iowa for technical supports. 6. Uchida S, Dimmeler S. Long noncoding RNAs in cardiovascular diseases. Circ Res. 2015;116:737-750. 7. Kornfeld JW, Bruning JC. Regulation of metabolism by long, non-coding RNAs. Front Genet. 2014;5:57. Abbreviations LncRNA: long non-coding RNA; DN: diabetic nephropathy; H3K4me1: histone H3 lysine 4 monomethylation; H3K4me3: histone H3 lysine 4 tri- methylation; WGCNA: weighted gene co-expression network analysis algorithm; TSS: transcription start sites; FPKM: fragments Per Kilobase of transcripts per Million mapped reads; FACS: fluorescence-activated cell sorting. 1. Atkins RC, Zimmet P. Diabetic kidney disease: act now or pay later. Kidney Int. 2010;77:375-377. 2. Vallon V, Platt KA, Cunard R, Schroth J, Whaley J, Thomson SC, Koepsell H, Rieg T. SGLT2 mediates glucose reabsorption in the early proximal tubule. J Am Soc Nephrol. 2011;22:104-112. 3. Sallstrom J, Carlsson PO, Fredholm BB, Larsson E, Persson AE, Palm F. Diabetes-induced hyperfiltration in adenosine A(1)-receptor deficient mice lacking the tubuloglomerular feedback mechanism. Acta Physiol. 2007;190:253-259. 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Zeng X, Lin W, Guo M, Zou Q. A comprehensive overview and evaluation of circular RNA detection tools. PLoS Comput Biol. 2017;13:e1005420. This work was supported by the NSFC (grant no. 81270805, 61502363, 61772394), and the department of science and technology of Sichuan province grant 2012FZ0076. 10. Zou Q, Li J, Song L, Zeng X, Wang G. Similarity computation strategies in the microRNA-disease network: a survey. Brief Funct Genomics. 2016;15:55-64. 11. Liu Y, Zeng X, He Z, Zou Q. Inferring microRNA-disease associations by random walk on a heterogeneous network with multiple data sources. IEEE/ACM Trans Comput Biol Bioinform. 2017;4:905-915. The authors declare no competing interest. The authors declare no competing interest. CONFLICTS OF INTEREST 8. Geisler S, Coller J. RNA in unexpected places: long non- coding RNA functions in diverse cellular contexts. Nat Rev Mol Cell Biol. 2013;14:699-712. Consent for publication DN. At least four light spots were revealed in current study. 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https://openalex.org/W4205816847	https://link.springer.com/content/pdf/10.1007/s10457-021-00727-2.pdf	English	null	Certification of açaí agroforestry increases the conservation potential of the Amazonian tree flora	Agroforestry systems	2,022	cc-by	5,991	Agroforest Syst (2022) 96:407–416 https://doi.org/10.1007/s10457-021-00727-2 (0123456789().,-volV)( 01234567 89().,-volV) Gabriel Damasco . Mandy Anhalt . Ricardo O. Perdiz . Florian Wittmann . Rafael L. de Assis . Jochen Scho¨ngart . Maria T. F. Piedade . Christine D. Bacon . Alexandre Antonelli . Paul V. A. Fine Received: 1 June 2021 / Accepted: 20 December 2021 / Published online: 8 January 2022 The Author(s) 2022 such increase represents a ﬁnancial boom for local communities, unregulated extraction in Amazonia risks negative environmental effects including biodi- versity loss through ac¸ai intensiﬁcation and deforesta- tion. Alternatively, the introduction of certiﬁed sustainable agroforestry production programs of ac¸aı´ has been strategically applied to reduce the exploita- tion of Amazonian forests. Local ac¸aı´ producers are such increase represents a ﬁnancial boom for local communities, unregulated extraction in Amazonia risks negative environmental effects including biodi- versity loss through ac¸ai intensiﬁcation and deforesta- tion. Alternatively, the introduction of certiﬁed sustainable agroforestry production programs of ac¸aı´ has been strategically applied to reduce the exploita- tion of Amazonian forests. Local ac¸aı´ producers are Abstract The harvesting of ac¸aı´ berries (palm fruits from the genus Euterpe) in Amazonia has increased over the last 20 years due to a high local and global market demand and triggered by their widely acclaimed health beneﬁts as a ‘superfood’. Although Supplementary Information The online version contains supplementary material available at https://doi.org/10.1007/ s10457-021-00727-2. G. Damasco (&) C. D. Bacon A. Antonelli Department of Biological and Environmental Sciences, University of Gothenburg, Go¨teborg, Sweden e-mail: gabriel.damasco@ufrn.br; gabrielﬂoresta@gmail.com G. Damasco C. D. Bacon A. Antonelli Gothenburg Global Biodiversity Centre, Go¨teborg, Sweden G. Damasco Department of Botany and Zoology, Universidade Federal Do Rio Grande Do Norte, Natal, Brazil M. Anhalt Global Compliance Department, Sambazon, Inc, San Diego, CA, USA R. O. Perdiz F. Wittmann J. Scho¨ngart M. T. F. Piedade Instituto Nacional de Pesquisas da Amazoˆnia, Manaus, AM, Brazil R. O. Perdiz Luz da Floresta, Rua Xiriana, 311, Aparecida, Boa Vista, RR, Brazil Supplementary Information The online version contains supplementary material available at https://doi.org/10.1007/ s10457-021-00727-2. G. Damasco (&) C. D. Bacon A. Antonelli Department of Biological and Environmental Sciences, University of Gothenburg, Go¨teborg, Sweden e-mail: gabriel.damasco@ufrn.br; gabrielﬂoresta@gmail.com F. Wittmann Department of Wetland Ecology, Institute for Geography and Geoecology, Karlsruhe Institute for Technology, Karlsruhe, Brazil R. L. de Assis Natural History Museum, University of Oslo, Oslo, Norway G. Damasco C. D. Bacon A. Antonelli Gothenburg Global Biodiversity Centre, Go¨teborg, Sweden G. Damasco C. D. Bacon A. Antonelli Gothenburg Global Biodiversity Centre, Go¨teborg, Sweden A. Antonelli Royal Botanic Gardens Kew, Richmond, UK A. Antonelli Royal Botanic Gardens Kew, Richmond, UK G. Certiﬁcation of ac¸aı´ agroforestry increases the conservation potential of the Amazonian tree ﬂora Gabriel Damasco . Mandy Anhalt . Ricardo O. Perdiz . Florian Wittmann . Rafael L. de Assis . Jochen Scho¨ngart . Maria T. F. Piedade . Christine D. Bacon Alexandre Antonelli . Paul V. A. Fine Gabriel Damasco . Mandy Anhalt . Ricardo O. Perdiz . Florian Wittmann . Rafael L. de Assis . Jochen Scho¨ngart . Maria T. F. Piedade . Christine D. Bacon . Alexandre Antonelli . Paul V. A. Fine Damasco Department of Botany and Zoology, Universidade Federal Do Rio Grande Do Norte, Natal, Brazil A. Antonelli Department of Plant Sciences, University of Oxford, Oxford, UK P. V. A. Fine Department of Integrative Biology and Jepson and University Herbaria, University of California, Berkeley, CA 94720-3140, USA R. O. Perdiz F. Wittmann J. Scho¨ngart M. T. F. Piedade Instituto Nacional de Pesquisas da Amazoˆnia, Manaus, AM, Brazil R. O. Perdiz Luz da Floresta, Rua Xiriana, 311, Aparecida, Boa Vista, RR, Brazil R. O. Perdiz Luz da Floresta, Rua Xiriana, 311, Aparecida, Boa Vista, RR, Brazil 123 Agroforest Syst (2022) 96:407–416 408 required to follow principles of deﬁned sustainable management practices, environmental guidelines, and social behaviors, paying speciﬁc attention to fair trade and human rights. In this study we investigate whether sustainable agroforestry and certiﬁcation effectively promotes biodiversity conservation in Amazonia. To address this question, we conducted a forestry inven- tory in two hectares of long-term certiﬁed ac¸ai harvesting areas to gain further knowledge on the plant diversity and forest structure in ac¸aı´ managed forests and to understand the contribution of certiﬁ- cation towards sustainable forest management. On average, we found that certiﬁed managed forests harbor 50% more tree species than non-certiﬁed ac¸aı´ groves. Trees in certiﬁed areas also have signiﬁcantly higher mean basal area, meaning larger and hence older individuals are more likely to be protected. Certiﬁed harvesting sites also harbor dense popula- tions of threatened species as classiﬁed by the International Union for Conservation of Nature (e.g. Virola surinamensis, classiﬁed as ‘endangered’). Besides increasing the knowledge of plant diversity in ac¸aı´ managed areas, we present baseline information for monitoring the impact of harvesting activities in natural ecosystems in Amazonia. management leads to an erosion of the regional ﬂora and can cause considerable changes in the vegetation structure and ecosystem functioning of Amazonian forests (Freitas et al. 2015). Given the rampant habitat loss and reduction in forest biodiversity due to recent ac¸aı´ expansion (Freitas et al. 2015; Campbell et al. 2018; Bezerra et al. 2020) and the vulnerability of ac¸aı´ palm forests to climate change (Tregidgo et al. 2020), an innovative strategy for sustainable agroforestry management is urgently needed to prevent biodiversity loss and deforestation. Currently, the ac¸aı´ market in Brazil lacks a clear and uniform policy that regulates the promotion of biodiversity conservation in harvesting areas. Gabriel Damasco . Mandy Anhalt . Ricardo O. Perdiz . Florian Wittmann . Rafael L. de Assis . Jochen Scho¨ngart . Maria T. F. Piedade . Christine D. Bacon . Alexandre Antonelli . Paul V. A. Fine Yet, due to high demand for organic and sustainable products, ac¸aı´ food companies (especially from North America and Europe) have invested in certiﬁcation programs that inform consumers about whether the product is in accordance with health, ecosystem conservation, and human rights (e.g. USDA Organic, Regenerative Organic, Biodynamic, Fair trade and Fair for Life). Certiﬁed ac¸aı´ agroforestry may provide many environmental and social beneﬁts. It can improve livelihoods for local residents, promote conservation of intact forests that harbor high biodiversity, and prevent unregulated deforestation that, in turn, exac- erbates global climate change (Weinstein and Moe- genburg 2004; Lovejoy and Nobre 2018; Brando et al. 2020). Certiﬁers have a diverse array of objectives and standards, meaning that they do not necessarily address all facets of sustainability (e.g., Perrigo et al. 2020), but most deﬁne criteria relating to environ- mental and social outcomes (World Conservation Monitoring Centre 2011). Keywords Euterpe Ac¸aı´ management Flooded forests Biodiversity conservation Amazonia Introduction The expansion of the ac¸aı´ (Euterpe sp.) fruit economy in Amazonia has not only supplied a growing world- wide demand but has also helped to establish new frontiers of commercialization. The market boom for ac¸aı´ berries has motivated producers to expand ac¸aı´ agroforestry into pristine and unmanaged forests, especially in eastern Amazonia (Brondizio 2008). Although ac¸aı´ agroforestry does not involve outright deforestation, intensive management practices gradu- ally replace native tree species by thinning and/or with dense ac¸aı´ palm plantings in the understory to enhance productivity (Homma et al. 2006; Freitas et al. 2015). Besides, management practices typically include the gradual removal of other native trees near ac¸aı´ palms, promoting canopy opening, and reducing overall competition for light and nutrients. This unregulated In tropical countries, such as Brazil, third party certiﬁcation could play an important role considering the negative effects of a growing commodity produc- tion (Gibbs et al. 2010), and the need for governmental capacity and resources to regulate agriculture effec- tively (Barrett et al. 2001). ‘‘Fair for Life’’ is a Fair Trade certiﬁcation standard developed and managed by EcoCert Group. Sambazon Inc., has been certiﬁed to the ﬁrst standard developed by Ecocert since 2008 and was the ﬁrst company to have Fair Trade certiﬁed ac¸aı´. ‘‘Fair for Life’’ is composed by the ‘‘Fair Trade and Responsible supply-chain’’ certiﬁcation and the ‘‘For Life Standard’’ for ‘‘Corporate Social Responsi- bility’’ certiﬁcation. Both have environmental 123 Agroforest Syst (2022) 96:407–416 409 12 years through the partnership between Sambazon Inc. (https://www.sambazon.com/) and ECOCERT Group (https://www.ecocert.com) as part of the ‘‘Fair for Life’’ fair trade certiﬁcation. This certiﬁcation is based on principle that threatened or endangered species and habitats are protected and natural ecosystems are not destroyed. More information about requirements and criteria that are expected to be met under the Fair for Life certiﬁcation is available in the Table S1. requirements at the local and corporate levels as a corner stone of the certiﬁcation scheme. For an ac¸aı´ product to become Fair for Life certiﬁed, local producers and harvesters must follow standards regarding the respect of human rights and fair working conditions, promotion of biodiversity and sustainable agroforestry practices, and have a positive impact on local communities (EcoCert Group 2020). Each operation is audited annually by the Ecocert group to ensure the companies are following these rules. Introduction With the increasing public, governmental, and corporate interest in sustainable consumption (Govin- dan 2018; Matharu et al. 2020), it is important to understand the contribution of sustainable agro- forestry and certiﬁcation in promoting biodiversity conservation. Here, we determine tree species com- position and forest structure of fair for life certiﬁed ac¸aı´ harvesting areas in eastern Amazonia and com- pare our results with (1) non-certiﬁed ac¸aı´ plantations and (2) transects in intact Amazonian ﬂooded forests. Our aim is to explore the potential contribution of ac¸aı´ agroforestry and certiﬁcation to halting deforestation and species loss in Amazonian forests. We predict that certiﬁed harvesting areas harbor a richer tree commu- nity and a less impacted forest than non-certiﬁed sites. Additionally, this study establishes research plots in areas never sampled before and used sampling methodologies compatible with protocols used by researchers worldwide. Thus, our results could poten- tially contribute to larger-scale biodiversity studies. This study comprises three main distinct sets of inventory plots: (1) 20 plots of certiﬁed ac¸aı´ managed forests established along in eastern Amazonia along the Mariazinho River in Breves County, Para´ State, Brazil (S 00 51.9020 W 51 04.9610) and Gurupa´ County, Para´ State, along the Mararu´ River (S 1 11.1670 W 51 32.7320) and in the Bailique Archipelago region, Amapa´ State (N 028049.7700 and W 5024039.7400); 2) 24 plots of non-certiﬁed ac¸aı´ groves published by Freitas et al. (2015) named in their publication as ‘‘managed sites’’; (3) total of 50 plots from intact and unmanaged ﬂooded forests sites (named here as control plots). 38 control plots were made available by the PELD-MAUA research group (https://peld-maua.inpa.gov.br). This research net- work manages a large dataset of forestry inventories held across ﬂooded forests in the Amazon Basin (Fig. 1, Table S3). In addition, 12 control plots were published by Freitas et al. (2015), named in their publication as ‘‘unmanaged control sites’’ (Fig. 1, Table S3). Sampling Study sites For certiﬁed harvesting areas, the 20 plots were 0.1 ha in size each (20 m 9 50 m), representing a cumula- tive sampling area of 2 hectares. Using maps and GIS resources, the 0.1 ha-plots were randomly selected a priori and established within each of the twenty certiﬁed harvesting lands visited. All woody plant species with a diameter at the breast height (DBH) above 10 cm (considered here as ‘trees’) were mea- sured, collected, and identiﬁed to species when possible. The ﬁeldwork was comprised by two expe- ditions held in August 2019 and January 2020. Voucher specimens for each tree species have been deposited at the INPA (Instituto Nacional de Pesquisas da Amazoˆnia) Herbarium in Manaus, Brazil. Taxo- nomic classiﬁcation followed APG IV (http://www. The certiﬁed harvesting areas sampled in our study are located in Eastern Amazonia. Sampling sites were placed on private lands situated along tributary water channels of the Amazonas River. The primary habitat in this region is seasonally ﬂooded forest (known in Amazonia as va´rzea forests) and the ac¸aı´ palm (Euterpe oleracea Mart.) is a dominant and wide- spread palm species native to these habitats. Here, we conducted tree-plot inventories that encompassed the spectrum of ac¸aı´ management varying from an almost complete replacement of trees with ac¸aı´ to no tree removal or destructive interference. We sampled harvesting lands that have been certiﬁed in the last 12 3 3 Agroforest Syst (2022) 96:407–416 410 Fig. 1 Geographical location of 94 study cites located across ﬂooded forests in the Brazilian Amazonia. Control plots represent intact ﬂooded forests with no inﬂuence of ac¸aı´ management practices. 38 control plots were made available by the PELD-MAUA data network (https://peld-maua.inpa.gov. br) and 12 control plots are available in Freitas et al. (2015). Non-certiﬁed plots are available in Freitas et al. (2015) and comprise of forests managed for ac¸aı´ production by riverine smallholders that are free of certiﬁcation standards. Certiﬁed plots are made available by this study and represent forests managed for ac¸aı´ production that has been under the Fair for Life certiﬁcation since 2008 Non-certiﬁed plots are available in Freitas et al. (2015) and comprise of forests managed for ac¸aı´ production by riverine smallholders that are free of certiﬁcation standards. Certiﬁed plots are made available by this study and represent forests managed for ac¸aı´ production that has been under the Fair for Life certiﬁcation since 2008 Fig. Sampling 1 Geographical location of 94 study cites located across ﬂooded forests in the Brazilian Amazonia. Control plots represent intact ﬂooded forests with no inﬂuence of ac¸aı´ management practices. 38 control plots were made available by the PELD-MAUA data network (https://peld-maua.inpa.gov. br) and 12 control plots are available in Freitas et al. (2015). was calculated based on the formula g = (p 9 DBH2)/4 where g is basal area in m2. To compare the diversity and forest structure of certiﬁed, non-certiﬁed, and control plots we used the non-parametric Wilcoxon test as variables were not normally distributed. We then performed a linear model to test the association between species richness/basal area and the ac¸aı´ density among certiﬁed, non-certiﬁed, and control plots. All statistical analyses were performed in R 3.4.1 (R Core Team 2020) using the package ‘‘vegan’’ (Oksanen et al. 2020). mobot.org) and species names were checked against the Plants of the World online (POWO, http://www. plantsoftheworldonline.org/). For details of sampling methods of non-certiﬁed harvesting areas and control plots (Table S3), please see Freitas et al. (2015) methods section and the PELD-MAUA research group data repository (https://peld-maua.inpa.gov.br). Analysis We calculated diversity (species richness, Fisher diversity index) and forest structure (basal area, ac¸aı´ clump density) parameters for the certiﬁed, non- certiﬁed, and control data sets. Diversity and basal area metrics are calculated accordingly to scientiﬁc literature on Amazonian ﬂoodplains (e.g., Assis et al. 2015, 2019). Fisher’s a diversity index was deter- mined according to the formula S = a 9 ln (1 ? n/a) where S is the number of species, n is the number of individuals, and a is Fisher’s alpha. Tree basal area We acknowledge that the control, certiﬁed and non- certiﬁed plots are not evenly distributed across the geographic space and the majority of certiﬁed and non-certiﬁed plots are skewed to Eastern Amazonia. Therefore, results on tree diversity and forest structure could also reﬂect responses to geographic variation, including the climatic difference, disturbance history and the effects associated with the spatial distance across the region. Despite the potential spatial bias, 12 123 Agroforest Syst (2022) 96:407–416 411 diameters over 120 cm at the breast height (DBH). All certiﬁed studied plots obtained basal area values (26.9–91.7 m2.ha-1) within the range of variation comparable to intact forests (Table 1, Fig. 2). 123 411 diameters over 120 cm at the breast height (DBH). All certiﬁed studied plots obtained basal area values (26.9–91.7 m2.ha-1) within the range of variation there is no known evidence for local tree diversity, long-term disturbance history or climate in ﬂooded va´rzea forests to be remarkably different between certiﬁed plots vs. non-certiﬁed plot sites. In Eastern Amazonia, Amapa´ and Para´ States are two of the most expressive areas in terms of ac¸aı´ production and marketing in Brazil and ac¸aı´ managed forests are frequently found across va´rzea forests in this region. To disentangle the effects of certiﬁcation, climatic differences, and geographic distance among plots, we performed a decomposition of the explained variation using distance-based redundancy analysis (db-RDA) available in the R package ‘‘vegan’’ (Oksanen et al. 2020). Temperature and precipitation data were extracted from the Worldclim database (www. worldclim.org/data) and the variation of temperature and precipitation variables were reduced based on principal component analysis. Linear distances among all plots were calculated using the R package ‘‘geo- sphere’’ (Karney 2013). Results Species composition and structure of certiﬁed ac¸aı´ harvesting forests Species composition and structure of certiﬁed ac¸aı´ harvesting forests In total, we sampled 131 tree species at the certiﬁed sites represented by 36 botanical families and 109 genera (Table S2). The number of families and tree species accounted for 50% and 18% of the MAUA database (control group) that includes ﬂoristic data from forests across Amazonia. On average, certiﬁed ac¸aı´ managed forests harbored a greater number of tree species than non-certiﬁed ac¸aı´ (Fig. 2a). The number of tree species was signiﬁcantly higher (P = 0.00634) in certiﬁed harvesting areas and 50% higher than non- certiﬁed groves, on average (Table 1). In contrast, both certiﬁed and non-certiﬁed forests are less diverse in terms of species richness in comparison to the control plots (P = 1.0087e-9, P = 1.654e-8 for both pairwise Wilcoxon tests). In terms of forest structure, certiﬁed ac¸aı´ forests had trees with greater values of basal area per hectare (P = 0.01567e-6), Fig. 2). On average, basal area values were ca. 150% higher in certiﬁed forests (Table 1). The largest individual trees were from the species Ceiba pentandra (L.) Gaertn., Spondias mon- bin L. and Mora paraensis (Ducke) Ducke with diameters over 120 cm at the breast height (DBH). All certiﬁed studied plots obtained basal area values (26.9–91.7 m2.ha-1) within the range of variation comparable to intact forests (Table 1, Fig. 2). 12 3 Agroforest Syst (2022) 96:407–416 412 Table 1 Floristic and structural parameters of certiﬁed, non-certiﬁed ac¸aı´ harvesting areas and native va´rzea ﬂooded forests No. families Species richness Fisher’s a diversity Basal area (m2.ha-1) Ac¸aı´ clump density (ha-1) No. plots Control 24.4 ± 7 (39–12) 54.4 ± 20.8 (100–14) 20.3 ± 8.7 (39.2–5.22) 89.1 ± 30.5 (138.7–22.19) 25.4 ± 45.2 (210–0) 50 Non-certiﬁed 8 ± 2.6 (13–3) 12.6 ± 5.5 (26–3) 4.4 ± 2.7 (10.8–0.6) 22.8 ± 11.8 (40.4–8) 549.1 ± 136.7 (1040–270) 24 Certiﬁed 10.6 ± 2.8 (14–7) 17.6 ± 5.6 (30–8) 5.4 ± 2.8 (10.3–1.96) 58 ± 10.9 (91.7–26.9) 538.5 ± 203.9 (1200–90) 20 Control data represent the PELD-MAUA dataset, a tree-plot network for seasonally ﬂooded forests located across the Amazon Basin. Numbers displayed represent the average ± standard deviation (max min range) bFig. 2 Comparisons of tree species richness, basal area, and density of ac¸aı´ clumps among certiﬁed, non-certiﬁed and control plots. Boxplots above shows the distribution of the data based on the median, ﬁrst and third quartile, and minimum and maximum values. Species composition and structure of certiﬁed ac¸aı´ harvesting forests Letters on the left upper corner of each box represent the result of the pairwise and non-parametric Wilcoxon test. Distinct letters indicate a signiﬁcant statistical difference among groups (p-values are displayed in the main text of the manuscript) Table 1 Floristic and structural parameters of certiﬁed, non-certiﬁed ac¸aı´ harvesting areas and native va´rzea ﬂooded forests No. families Species richness Fisher’s a diversity Basal area (m2.ha-1) Ac¸aı´ clump density (ha-1) No. plots Control 24.4 ± 7 (39–12) 54.4 ± 20.8 (100–14) 20.3 ± 8.7 (39.2–5.22) 89.1 ± 30.5 (138.7–22.19) 25.4 ± 45.2 (210–0) 50 Non-certiﬁed 8 ± 2.6 (13–3) 12.6 ± 5.5 (26–3) 4.4 ± 2.7 (10.8–0.6) 22.8 ± 11.8 (40.4–8) 549.1 ± 136.7 (1040–270) 24 Certiﬁed 10.6 ± 2.8 (14–7) 17.6 ± 5.6 (30–8) 5.4 ± 2.8 (10.3–1.96) 58 ± 10.9 (91.7–26.9) 538.5 ± 203.9 (1200–90) 20 Control data represent the PELD-MAUA dataset, a tree-plot network for seasonally ﬂooded forests located across the Amazon Basin. Numbers displayed represent the average ± standard deviation (max–min range) Although species richness and basal area signiﬁ- cantly differed between certiﬁed and non-certiﬁed plots, we found no signiﬁcant difference between ac¸aı´ abundance when both groups are compared (Fig. 2c, P = 0.8781). Therefore, certiﬁed areas were richer in terms of ﬂoristic composition, had a larger forest stature (higher basal area), and possessed comparable ac¸aı´ density as non-certiﬁed forests. Relationship between richness and ac¸aı´ clump density We found a negative relationship between the density of ac¸aı´ clumps and the species richness in ac¸aı´ managed forests. The higher the ac¸aı´ clump density is, the lower the diversity of tree species is in both certiﬁed and non-certiﬁed harvesting areas (Fig. 3). However, certiﬁed data showed a trend with a lower slope than non-certiﬁed data (slopecertiﬁed = - 0.01,R2 certiﬁed = 0.215;slopenon-certiﬁed = - 22.75, R2 non-certiﬁed = 0.677, respectively). Even in sites with a high density of ac¸aı´ clumps, certiﬁed harvesting areas tend to have a more species rich composition and a larger tree basal area than non-certiﬁed ac¸aı´ groves (Table 1, Fig. 3). The relationship between the density of ac¸aı´ clumps and the species richness is still signiﬁcant when the explained variation is partitioned using the db-RDA analysis. Despite the climatic distinction over a large spatial range among the studied plots (Figure S1A), we did not ﬁnd supportive evidence that species richness could be associated with the climatic variation over the space. We did ﬁnd a partial association between richness and the spatial distances among the plots (Figure S1B), possibly due to the overwhelming number of control plots from the PELD-MAUA research network that are located in western Amazo- nia. Even though the spatial correlation is present, the number of species among certiﬁed, non-certiﬁed, and 123 123 Agroforest Syst (2022) 96:407–416 413 Fig. 3 Linear correlation between tree species richness and basal area against density of ac¸aı´ clumps in certiﬁed and non- certiﬁed plots. Both graphs show a negative relationship and as the density of ac¸aı´ clumps increases in managed forests the number of species and the basal area of both certiﬁed and non- certiﬁed tend to decrease. However, the decrease in species richness at certiﬁed plots is less pronounced than non-certiﬁed plots (slope and R2 values are displayed in the main text of the manuscript) Agroforest Syst (2022) 96:407–416 413 d by the d forests Euphorbiaceae (4 spp. 49 individuals). These families together account for more than 50% of the total number of tree species sampled in certiﬁed managed Fig. 3 Linear correlation between tree species richness and basal area against density of ac¸aı´ clumps in certiﬁed and non- certiﬁed plots. Both graphs show a negative relationship and as the density of ac¸aı´ clumps increases in managed forests the number of species and the basal area of both certiﬁed and non- certiﬁed tend to decrease. Relationship between richness and ac¸aı´ clump density However, the decrease in species richness at certiﬁed plots is less pronounced than non-certiﬁed plots (slope and R2 values are displayed in the main text of the manuscript) control plots was still signiﬁcantly explained by the density of ac¸aı´ clumps in the studied managed forests (Figure S1B). Euphorbiaceae (4 spp. 49 individuals). These families together account for more than 50% of the total number of tree species sampled in certiﬁed managed sites (Table S2). Virola surinamensis, the most representative tree species, and Minquartia guianensis are registered in the IUCN red list as endangered taxa. Some popular agroforestry species, including non- natives, were also found amid certiﬁed forests (e.g. Mangifera indica, Theobroma cacao, Spondias mom- bin, Musa spp.). Discussion Can certiﬁcation of ac¸aı´ agroforestry support conservation of the Amazonian ﬂora? Despite the lack of a temporal monitoring assessment in our experimental setup, we identify that managed sites after 12 years of certiﬁcation have a higher species richness than non-certiﬁed groves. Addition- ally, the forest structure of certiﬁed sites is consider- ably more intact compared to non-certiﬁed areas. Even though ac¸aı´ abundance is similar across the different sites and the potential for fruit productivity may be then equivalent, the basal area of native trees was much higher in certiﬁed than non-certiﬁed managed areas. We presume that certiﬁed producers preferen- tially take conservative and lower impact measures regardless of the prior state of conservation of managed sites. Therefore, even if the certiﬁed plots included in our study had a better conservation status (e.g., with higher species richness and biomass) before certiﬁcation was implemented, certiﬁed and trained producers acted differently than the intense and uncontrolled management usually applied by non- certiﬁed producers (as in Freitas et al. 2015). In addition, regardless of a possible geographical bias in the sampling design, certiﬁcation and best manage- ment practices on their own appear to have a noteworthy effect on protecting the tree diversity and the forest structure of ac¸aı´ harvesting forests. Our comparisons allow us to conclude that there is a high conservation potential of certiﬁed ac¸aı´ agroforestry to reduce the degradation of the Amazonian tree ﬂora. Recent studies have assessed the ecological out- comes and provided evidence for the positive impacts of certiﬁcation (Willemen et al. 2019; Furumo et al. 2020, Pico-Mendonza et al. 2020). For instance, certiﬁed coffee agroforestry from Costa Rica were less prone to deforestation and provided more ecosys- tem services than non-certiﬁed forests (Pico-Men- donza et al. 2020). Similar ﬁndings were shown in Colombia where certiﬁed coffee plantations had an increased diversity of shade-trees compared to uncer- tiﬁed farms (Ibanez and Blackman 2016). However, certiﬁcation also has its limitations (Waldman and Kerr 2014), with complex and expensive processes that often limit small productions (Loconto and Dankers 2014; Brandi et al. 2015). Although certiﬁ- cation is not the only avenue to sustainability, it can provide a regulated system to accomplish and detail advancements through identifying indicators and auditing ac¸aı´ management practices. In line with our results, we suggest that ac¸aı´ certiﬁcation can promote ecologically sound manage- ment to supply national and international markets. Flora of certiﬁed ac¸aı´ managed forests Flora of certiﬁed ac¸aı´ managed forests The most dominant botanical families found in the certiﬁed plots were Fabaceae (27 spp., 303 individu- als), followed by Myristicaceae (2 spp., 95 individu- als), Malvaceae (9 spp., 78 individuals), Anacardiaceae (2 spp., 64 individuals), and 123 414 Agroforest Syst (2022) 96:407–416 higher basal area. Surprisingly, ac¸aı´ density between certiﬁed and non-certiﬁed managed sites was not signiﬁcantly different. Therefore, certiﬁed managed sites can hypothetically maintain fruit production at similar rates to non-certiﬁed forests, if the mean fruit production per ac¸aı´ palm happens to be the same disregarding the abiotic differences among plots. Discussion In addition, with a combined effort to aggregate producer cooperatives, certiﬁcation provides a platform to better support the livelihoods of ac¸aı´ producers as they face the growing and changing market. During the certiﬁcation training, producers are informed about the role of biodiversity and respective ecosystem services. In addition, harvesters are pro- hibited from engaging in illegal logging and are encouraged to avoid excessive cutting of large trees at managed sites. Although less destructive than clear- cutting of tropical forests, unregulated ac¸aı´ manage- ment can still cause considerable losses to the species composition and forest structure of Amazonian forests. For instance, certiﬁed managed sites harbor many trees with large diameters (larger than 100–120 cm of DBH) and heights (30–40 m). Accord- ing to the relationship between richness/biomass and ac¸aı´ density described by Freitas et al. (2015), we expected to ﬁnd low values of ac¸aı´ density at the certiﬁed study sites, especially in those plots with References Anderson AB, Jardim MAG (2019) Costs and beneﬁts of ﬂoodplain forest management by rural inhabitants in the Amazon estuary: a case study of Ac¸aı´ palm production. In: Fragile lands of Latin America. Routledge, pp 114–129 Assis RL, Haugaasen T, Scho¨ngart J, Montero JC, Piedade MT, Wittmann F (2015) Patterns of tree diversity and compo- sition in Amazonian ﬂoodplain paleo-va´rzea forest. J Veg Sci 26(2):312–322 Embracing a tactical approach for expanding certiﬁcation in priority zones in Amazonia should involve collaboration across a number of sectors. Governments and civil society will have to act along with ac¸aı´ food companies and investors to identify how certiﬁcation might offer the greatest beneﬁts to biodiversity protection and sustainable livelihoods. Frontiers of expansion for commercial ac¸aı´ groves could be the focal point for the application of norms to safeguard intact forests. Assis RL, Wittmann F, Bredin YK, Scho¨ngart J, Quesada CAN, Piedade MTF, Haugaasen T (2019) Above-ground woody biomass distribution in Amazonian ﬂoodplain forests: effects of hydroperiod and substrate properties. For Ecol Manage 432:365–375 Barrett CB (2001) Measuring integration and efﬁciency in international agricultural markets. Appl Econ Perspect Policy 23(1):19–32 Bezerra LA, Campbell AJ, Brito TF, Menezes C, Maue´s MM (2020) Pollen loads of ﬂower visitors to ac¸aı´ palm (Euterpe oleracea) and implications for management of pollination services. Neotrop Entomol 49(4):482–490 Brandi C, Cabani T, Hosang C, Schirmbeck S, Westermann L, Wiese H (2015) Sustainability standards for palm oil: challenges for smallholder certiﬁcation under the RSPO. J Environ Dev 24(3):292–314 Authors’ contribution GD, MA and PVAF conceived the project. GD, PVAF and ROP performed ﬁeldwork and data collection. GD performed data analysis and led the manuscript writing. FW, RLA, JS and MTFP provided additional data from control plots and revised the manuscript. AA, CDB and PVAF revised and supervised the manuscript writing. Brando PM, Soares-Filho B, Rodrigues L, Assunc¸a˜o A, Morton D, Tuchschneider D, Fernandes ECM, Macedo MN, Oli- veira U, Coe MT (2020) The gathering ﬁrestorm in southern Amazonia. Sci Adv 6(2):eaay1632 Funding Open access funding provided by University of Gothenburg. GD is funded by Vinnova (2019-02717) and Sambazon Inc. Biodiversity Grant. AA ifs funded by the Swedish Research Council, the Swedish Foundation for Strategic Research and the Royal Botanic Gardens, Kew. Brondı´zio ES (2008) The Amazonian Caboclo and the Ac¸aı´ palm: forest farmers in the global market. The future of ac¸aı´ management certiﬁcation The future of ac¸aı´ management certiﬁcation Considering the rising market, certiﬁcation can be a vital mechanism for gaining appreciation and increas- ing value for ac¸aı´ superfood products (Hogarth 2004; de Oliveira and Schwartz 2018). In 2016, approx- imetly 300,000 tons of ac¸aı´-based products were sold around the world (Future Market Insights 2017). Also, the ac¸aı´ global market is projected to exceed 1 million tons in sales and hit nearly 2 billion USD in revenue by the end of 2026 (Future Market Insights 2017). With more than 55% market share, Latin America will dominate the global sales towards the end of the projected period. 123 Agroforest Syst (2022) 96:407–416 415 In Brazil, new opportunities for ac¸aı´ management certiﬁcation are emerging (Anderson and Jardim 2019) and 92% of producers who had heard of certiﬁcation consider it an advantageous route for reaching a better valuation for their ac¸aı´ (Pepper and Alves 2017). Several third-party certiﬁcation pro- grams offered in Para´ and Amapa´ States (e.g. ECOCERT Brasil, IBD Certiﬁcations, and IMA- FLORA) are mostly directed to large-scale producers or cooperative farmers but these companies have started to extend programs more suitable for small- scale ac¸aı´ producers (Pepper and Alves 2017; Johnson et al. 2018), offering them an edge in international markets. included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. 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https://openalex.org/W2951777278	https://europepmc.org/articles/pmc6639725?pdf=render	English	null	Exploration of Multiple Signaling Pathways Through Which Sodium Tanshinone IIA Sulfonate Attenuates Pathologic Remodeling Experimental Infarction	Frontiers in pharmacology	2,019	cc-by	10,694	ORIGINAL RESEARCH doi: 10.3389/fphar.2019.00779 published: 12 July 2019 ORIGINAL RESEARCH doi: 10.3389/fphar.2019.00779 published: 12 July 2019 ORIGINAL RESEARCH doi: 10.3389/fphar.2019.00779 published: 12 July 2019 Exploration of Multiple Signaling Pathways Through Which Sodium Tanshinone IIA Sulfonate Attenuates Pathologic Remodeling Experimental Infarction Shuai Mao 1,2, Matthew Vincent 3, Maosheng Chen 1,2, Minzhou Zhang 1,2* and Aleksander Hinek 4 Shuai Mao 1,2, Matthew Vincent 3, Maosheng Chen 1,2, Minzhou Zhang 1,2* and Aleksander Hinek 4 1 Key Discipline of Integrated Traditional Chinese and Western Medicine, Second Clinical College, Guangzhou University of Chinese Medicine, Guangzhou, China, 2 Department of Critical Care Medicine, Guangdong Provincial Hospital of Chinese Medicine, Guangzhou, China, 3 Medical School, St. George’s, University of London, London, United Kingdom, 4 Physiology & Experimental Medicine, Hospital for Sick Children, Toronto, ON, Canada Edited by: Sanjoy Ghosh, University of British Columbia Okanagan, Canada The level of maladaptive myocardial remodeling consistently contributes to the poor prognosis of patients following a myocardial infarction (MI). In this study, we investigated whether and how sodium tanshinone IIA sulfonate (STS) would attenuate the post- infarct cardiac remodeling in mice model of MI developing after surgical ligation of the left coronary artery. All mice subjected to experimental MI or to the sham procedure were then treated for the following 4 weeks, either with STS or with a vehicle alone. Results of our studies indicated that STS treatment of MI mice prevented the left ventricular dilatation and improved their cardiac function. Results of further tests, aimed at mechanistic explanation of the beneficial effects of STS, indicated that treatment with this compound enhanced the autophagy and, at the same time, inhibited apoptosis of the cardiomyocytes. Meaningfully, we have also established that myocardium of STS-treated mice displayed significantly higher levels of adenosine monophosphate kinase than their untreated counterparts and that this effect additionally associated with the significantly diminished activities of apoptotic promoters: mammalian target of rapamycin and P70S6 kinase. Moreover, we also found that additional administration of the adenosine monophosphate kinase inhibitor (compound C) or autophagy inhibitor (chloroquine) practically eliminated the observed beneficial effects of STS. In conclusion, we suggest that the described multistage mechanism triggered by STS treatment enhanced autophagy, thereby attenuating pathologic remodeling of the post-infarct hearts. Reviewed by: Jiayu Ye, University of British Columbia Okanagan, Canada Xin Tu, Huazhong University of Science and Technology, China Correspondence: Minzhou Zhang minzhouzhang@aliyun.com Specialty section: This article was submitted to Ethnopharmacology, a section of the journal Frontiers in Pharmacology Received: 18 September 2018 Accepted: 17 June 2019 Published: 12 July 2019 Keywords: sodium tanshinone IIA sulfonate, cardiac remodeling, autophagy, apoptosis, AMP kinase pathway ORIGINAL RESEARCH doi: 10.3389/fphar.2019.00779 published: 12 July 2019 INTRODUCTION The maladaptive cardiac remodeling that often develops after myocardial infarction (MI) triggers adverse cardiovascular events that often lead to the ultimate heart failure if not properly treated (Daubert et al., 2015). Unfortunately, the pathological remodeling of the infarcted myocardium still develops in many MI patients, even those treated, with coronary angioplasty–reperfusion and standard drugs (Springeling et al., 2013). Therefore, post-MI remodeling represents an attractive target for new pharmacological approaches aimed at the improvement of morbidity and mortality in patients following MI. Results of previously published analyses of the post-MI myocardium strongly suggested that a rapid autophagy of the dead cells remnants constitutes an essential step in cardiac recovery both in human clinic and in experimental conditions (French et al., 2010). Meaningfully, the upregulation of autophagy observed in rapidly recovered MI patients associated with inhibition of their left ventricular (LV) dilatation during the evolution of LV hypertrophy decreased the rate of the heart failure following MI (Guo et al., 2012). Moreover, it has been shown that pharmacologic inhibition of the mechanistic target of rapamycin (mTOR) pathway, known to be centrally involved in pathological remodeling of the heart (with rapamycin), prevented the LV remodeling and limited the size of the experimental infarct (McMullen et al., 2004a; Buss et al., 2009). In contrast, it has been also established that experimental inhibition of the autophagy-associated Atg5 gene in the heart contributed to an accumulation of partially degraded dead cells remnants that associated with development of myocardial hypertrophy. Interestingly, in a mouse model of ischemia and reperfusion cardiac injury, the heightened autophagy was linked to a parallel promotion of survival of the hypoxic cardiomyocytes via the upregulation of the adenosine monophosphate (AMP) kinase- dependent pathway (Matsui et al., 2007). Inspired by those studies, we conducted the clinical trials— STS for reduction of periprocedural myocardial injury during percutaneous coronary intervention and STS for reduction of periprocedural myocardial injury during percutaneous coronary intervention remodeling (STS in LV remodeling secondary to acute MI) trials—to assess the effectiveness of STS on the prognosis of patients following MI (Mao et al., 2015a; Mao et al., 2015b). The obtained results have demonstrated a significant improvement in cardiac systolic/diastolic function and decreased incidence of major adverse cardiac events in patients treated with STS after revascularization. Citation: Mao S, Vincent M, Chen M, Zhang M and Hinek A (2019) Exploration of Multiple Signaling Pathways Through Which Sodium Tanshinone IIA Sulfonate Attenuates Pathologic Remodeling Experimental Infarction. Front. Pharmacol. 10:779. doi: 10.3389/fphar.2019.00779 July 2019 \| Volume 10 \| Article 779 Frontiers in Pharmacology \| www.frontiersin.org 1 STS Attenuates Pathologic Cardiac Remodeling Mao et al. Sodium tanshinone IIA sulfonate (STS, Figure 1), a derivative of tanshinone IIA, has been widely utilized in the clinical setting for its certified induction in alleviating symptoms of MI, preventing of thrombotic events, and improving of angina symptoms (Qiu et al., 2012). Results of the experimental in vivo studies demonstrated that the administration of STS ameliorated coronary no-reflow phenomenon and prevented microvascular obstruction through suppressing fibrinogen-like protein 2 expression and inflammation (Long et al., 2015). STS has also been shown to attenuate cardiac dysfunction, upregulate antioxidant systems, and lower the levels of circulating lipids in rats with isoproterenol-induced MI. These effects of STS treatment have been linked to its ability to upregulate the AMP kinase/acetyl CoA carboxylase pathways (Wei et al., 2013). Yet, another study demonstrated that STS could reduce oxidative stress-mediated apoptosis through inhibiting c-Jun N-terminal kinase activation, an enzyme known to play a critical role in cardiac remodeling (Yang et al., 2008). INTRODUCTION t Although a growing body of evidence indicated STS exerting a broad range of cardiovascular benefits, there have been no reports on its effect in post-infarction cardiac remodeling, especially in modulation of the balance between autophagy and apoptosis of cardiomyocytes in this process. Therefore, in the present study, in which the experimental MI was generated in mice by ligating their left anterior descending coronary artery, we investigated the possible beneficial actions of STS on post- infarct LV remodeling and subsequently to clarify the related molecular mechanisms. In recent years, tanshinone IIA, a potent pharmacological compound extracted from Salvia miltiorrhizabunge, received a great deal of attention due to the discovery of its effectiveness of anti-oxidant, anti-inflammatory, and anti-proliferative effects (Figure 1) (Wu et al., 2013; Mao et al., 2014; Hu et al., 2015). FIGURE 1 \| The chemical structures of tanshinone IIA and sodium tanshinone IIA sulfonate (STS). A sulfonate (STS). FIGURE 1 \| The chemical structures of tanshinone IIA and sodium tanshinone IIA sulfonate (STS). FIGURE 1 \| The chemical structures of tanshinone IIA and sodium tanshinone IIA sulfonate (STS). July 2019 \| Volume 10 \| Article 779 Frontiers in Pharmacology \| www.frontiersin.org Frontiers in Pharmacology \| www.frontiersin.org 2 STS Attenuates Pathologic Cardiac Remodeling Mao et al. MATERIALS AND METHODS set according to the clinical dose and previous reports in order not to cause adverse effects in the rodent heart (Zhou et al., 1999; Iwai-Kanai et al., 2008). To assess the effects of these treatments on mice without infarction, sham-operated mice were assigned to the same groups 4 weeks after surgery (n = 12 each). Echocardiographic Measurement Echocardiographic Measurement LV function and volumes were assessed with echocardiography using a Vevo 770 echocardiography system (Visual Sonics, Toronto, Canada). Briefly, mice were anesthetized with isoflurane/oxygen inhalation; once the short-axis two-dimensional image of the left ventricle was obtained at the papillary muscle level, two-dimensional guided M-mode images crossing the anterior and posterior walls were recorded. Diastolic thickness of the LV posterior wall and the inner dimension of the left ventricles in diastole or systole were measured in M-mode. Fraction shortening (FS), end-diastolic volume, and end-systolic volume were calculated as described previously (Qi et al., 2017). Assessment of cardiac output was done by echocardiography using Doppler. Estimation of stroke volume is done by the measuring of a diameter of a cardiac structure to assess the cross‐sectional area and multiplying it by the velocity time integral obtained by the Doppler. The cardiac output is calculated by multiplying stroke volume by heart rate. Histological Examination g Following echocardiographic measurements, the hearts were removed, weighed, then cut into halves longitudinally. One half specimen was fixed in 4% paraformaldehyde overnight, embedded in paraffin, cut into sections, and then stained with hematoxylin– eosin or Masson’s trichrome. Mean values of cardiomyocytes in the hematoxylin–eosin-stained LV cross sections from each mouse were calculated from 50 to 80 cells using microscopy at 400× magnification. Masson’s trichrome-stained sections were quantitatively analyzed using a light microscope at 40× magnification and a color image analyzer (QWin Colour Binary 1, LEICA) to evaluate myocardial fibrosis (blue fibrotic area as opposed to red myocardium). Animalsh The experimental procedures were carried out in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Academy of Sciences (8th edition, Washington DC, 2011) and were approved by the Institutional Animal Care and Use Committee of Guangdong Province Hospital of Chinese Medicine, Guangzhou University of Traditional Chinese Medicine. Wild-type C57BL/6 J mice (10–12 weeks old, male, 30 ± 5 g body weight) were obtained from the Experimental Animal Center of Guangdong Province. Materials STS was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China), and the contents were ≥98% by high-performance liquid chromatography. Chloroquine and compound C were purchased from Sigma- Aldrich (St. Louis, Missouri, USA). Anti-p62 antibody, anti-AMP kinase (AMPK) antibody, anti-phosphorylated AMPK antibody (Thr172), anti-P70S6K antibody, anti-phosphorylated P70S6K antibody (Thr309), anti-mTOR antibody, anti-phosphorylated mTOR antibody (Ser2448), and anti-myoglobin antibody were purchased from Cell Signaling Technology (Danvers, MA, USA). ATP bioluminescent assay kit was purchased from TOYO Inc. (Tokyo, Japan). Anti-atrial natriuretic peptide (ANP) antibody, anti-tubulin antibody, and fluorescein-conjugated goat anti-rabbit and fluorescein-conjugated goat anti-mouse were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-active caspase-3 antibody was purchased from Abcam Plc. (Cambridge, MA, USA). RNAspin Mini Kit used for isolating total RNA was purchased from GE Healthcare (Buckinghamshire, UK). All other chemicals used were of the highest grade available commercially. Sodium Tanshinone IIA Sulfonate Improved Cardiac Function in Mice After Myocardial Infarction Echocardiography was performed to evaluate the cardiac function of post-MI mice after 4 weeks of their respective treatments. Mice from the vehicle group exhibited significantly impaired cardiac function and diminished cardiac performance indices, including lower LV ejection fraction, fractional shortening (FS), and increased LV end-systolic diameter, and end-diastolic diameter (Figure 2, Supplementary Table 1). In the treatment group, STS was able to block the LV dilation, dysfunction, and the increase in LV end-diastolic volumes seen in the vehicle group. Meaningfully, the group treated with STS along with chloroquine showed cardiac dysfunction similar to that in the vehicle group (Figure 2, Supplementary Table 1). Experimental Protocols MI was generated in mice by ligating the left anterior descending coronary artery as previously described (Mao et al., 2018). In brief, after anesthetized and mechanically ventilated, the left anterior descending coronary artery of mice was exposed by thoracotomy and subsequent pericardiotomy. 8-0 silk suture was used to ligate artery, and then, MI was confirmed by ST segment elevation or the new emerged pathological Q waves on electrocardiogram using BL-420S Experiment System of Biological Function (TME, Shanghai, China). Mice in sham group were given the same surgical procedure without left anterior descending artery ligation. Three days after the procedure, the surviving mice were randomly divided into six groups assigned to one of the following treatments after echocardiographic examination: vehicle (vehicle, n = 20); 10 mg/kg per day of STS injected through peritoneal cavity (STS, n = 20); STS plus 10 mg/kg chloroquine injected through peritoneal cavity 30 min prior to STS administration (STS + Cq, n = 20); STS plus 20 mg/kg compound C injected through peritoneal cavity 30 min prior to STS administration (STS + CC, n = 20); and intraperitoneal 10 mg/kg chloroquine or 10 mg/kg compound C alone (n = 10, respectively). Each treatment was administered for 4 weeks. The doses of STS, chloroquine, and compound C were Immunohistochemistry and Quantification After paraffin removal, the sections were incubated with a primary antibody against microtubule-associated LC3 (MBL International, Woburn, MA). To observe the autophagic activity in cardiomyocytes, sections immunostained with anti-LC3 followed by Alexa 568 (red; Molecular Probes) were also labeled with anti- myoglobin antibody (DAKO Japan, Kyoto, Japan) followed by Alexa 488 (green; Molecular Probes, Sunnyvale, CA). These sections were then counterstained with Hoechst 33342 and observed under Nikon Eclipse E1000 microscope and Nikon Digital Sight Camera (Nikon Instruments, Tokyo, Japan). For in situ terminal deoxyuridine triphosphate nick end- labeling (TUNEL), tissue sections were stained with Fluorescein- FragEL (Oncogene Research Products, Boston, MA). Quantitative assessments were performed in 50 randomly chosen high-power fields (600) using an NIS-Element imaging software (Nikon Instruments, Tokyo, Japan). July 2019 \| Volume 10 \| Article 779 Frontiers in Pharmacology \| www.frontiersin.org 3 STS Attenuates Pathologic Cardiac Remodeling Mao et al. Electron Microscopyh (Invitrogen, USA) following the manufacturer’s protocol. PCR reaction was carried out in a total reaction volume of 15 µl, including: 7.5 µl of SYBR® Green PCR master mix (Applied Biosystems, CA), 5 ng of cDNA template, and 0.2 µM of gene-specific primers. Real- time PCR was carried out using an ABI Prism 7900HT (Applied Biosystems, Foster City, CA). The cycling conditions were: 95°C for 10 min, (95°C for 15 s, 60°C, 72°C for 1 min) for 40 cycles, followed by dissociation curve analysis. Changes in mRNA expression in ANP was normalized to 18s mRNA levels and compared statically (ΔΔCt method) using the ABI Prism SDS 2.1 software. The border zone of the ischemic heart tissue was quickly cut into 1 mm3 fractions, immersion fixed in 2.5% glutaraldehyde in 0.1 mol/l phosphate buffer overnight, and postfixed in 1% osmium tetroxide. The fractions were then dehydrated through an ascending series of alcohols and embedded in epoxy resin. The 90-nm ultrathin sections double-stained with uranyl acetate and lead citrate were routinely examined in an electron microscope (Hitachi H-600, Tokyo, Japan). Statistical Analysis y Forty microgram of the proteins extracted from hearts were resuspended in sample buffer [0.5 M Tris (hydroxymethyl) aminomethane hydrochloride, pH 6.8, 10% sodium dodecyl sulfate, 10% glycerol, 4% 2-β-mercaptoethanol, and 0.05% bromphenol blue] and boiled for 5 min. The protein lysates were resolved by 8–15% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to a polyvinylidenedifluroide membrane. The membranes were then probed using primary antibodies against LC3, p62, ANP, Sirt1, and AMPK; phosphorylated AMPK; P70S6 kinase; phosphorylated P70 S6 kinase; mTOR; or phosphorylated mTOR overnight at 4°C. The blots were rinsed and subsequently incubated in horseradish peroxidase-conjugated secondary antibodies in 5% bovine serum albumin for 1 h and developed using a chemiluminescent substrate. Protein expression or phosphorylation of immunodetected signaling molecules was quantified by densitometry using Quantity one software (Bio- Rad, California, USA). Tubulin served as the loading control. Data are expressed as means ± SEM. The significance of differences between groups was evaluated using one-way analysis of variance (ANOVA) with a post hoc Newman–Keuls multiple comparisons test or a repeated measures ANOVA with Bonferroni post hoc test. Values of P < 0.05 were considered significant. Quantitative Real-Time Reverse- Transcription Polymerase Chain Reaction Analysis Total RNA from hearts was prepared using the RNAspin Mini Kit (GE Healthcare). Reverse transcription of RNA was performed using SuperScript™ II Reverse Transcriptase with random primers FIGURE 2 \| STS improved echocardiographic parameters in mice after myocardial infarction (MI). Representative echocardiography pictures of M mode images (upper panels) and quantitative analysis (lower panels) of left ventricular fractional shortening (LVFS), LV ejection fraction (LVEF), LV end-systolic diameter (LVESD), and LV end-diastole diameter (LVEDD) were shown. #P < 0.05 versus sham; P < 0.05 versus vehicle; &P < 0.05 versus STS. Veh, vehicle; STS, sodium tanshinone IIA sulfonate; Cq, chloroquine. FIGURE 2 \| STS improved echocardiographic parameters in mice after myocardial infarction (MI). Representative echocardiography pictures of M mode images (upper panels) and quantitative analysis (lower panels) of left ventricular fractional shortening (LVFS), LV ejection fraction (LVEF), LV end-systolic diameter (LVESD), and LV end-diastole diameter (LVEDD) were shown. #P < 0.05 versus sham; P < 0.05 versus vehicle; &P < 0.05 versus STS. Veh, vehicle; STS, sodium tanshinone IIA sulfonate; Cq, chloroquine. July 2019 \| Volume 10 \| Article 779 Frontiers in Pharmacology \| www.frontiersin.org Mao et al. STS Attenuates Pathologic Cardiac Remodeling exposure to STS significantly limited the maladaptive myocardial remodeling induced by the experimental MI (Figure 3). Importantly, neither STS nor chloroquine affected LV geometry or cardiac function in the sham-operated heart, suggesting that STS did not affect cardiac function under non-pathological conditions. Importantly, we have also established that the post-MI mice that were treated with combination of STS and chloroquine developed similar rates of pathologic LV dilation and fibrosis as their vehicle-treated counterparts (Figures 3A, C, D). Sodium Tanshinone IIA Sulfonate Reversed Cardiac Remodeling in Mice After Myocardial Infarction p g Previous studies have shown that increased levels of the atrial natriuretic peptide (ANP) or B-type natriuretic peptide (BNP) closely correlate with the severity of post-infarct LV dysfunction. Meaningfully, our Western blot and qRT-PCR analyses showed that hearts of mice with experimental MI that were vehicle- treated exhibited significantly higher levels of ANP or BNP message and protein than sham-operated controls and that such abnormal MI-induced increase was completely attenuated in mice treatment with STS. Interestingly, when mice were treated with combination of STS and chloroquine, the levels of ANP or BNP resembled those seen in vehicle-treated controls (Figures 3E, F and Supplementary Figure A). Due to the prompt remodeling of the MI-injured hearts that usually involve both, degradation of the existing and production of the new extracellular matrix, the hearts adopted a more spherical shape. More precisely, we observed that all hearts of mice that were vehicle-treated for 4 weeks after experimental MI demonstrated a progressive dilatation of their LVs (Figure 3A). In contrast, hearts of MI-injured mice from the parallel, STS-treated group, showed only minute LV dilatation, when compared with the sham-operated mice than those from the vehicle group.f Moreover, along with its effects on LV cavity geometry, treatment with STS also remarkably decreased the ratio of heart weight to whole-body weight and reduced the areas occupied by the interstitial myo-fibroblasts and fibrotic tissue in the post MI scars (Figures 3B–D), when compared with the vehicle-treated group. These observations suggested that the 4-week-long Sodium Tanshinone IIA Sulfonate Amplified Autophagy in the Post-Infarct Heartsf investigated whether levels of apoptosis detectable in the border zones of infarcted hearts might be modified by STS treatment. investigated whether levels of apoptosis detectable in the border zones of infarcted hearts might be modified by STS treatment. i We were particularly eager to establish whether the overall beneficial activity of STS might be due to inhibition of apoptosis of cardiomyocytes located in the border zones of infarcted hearts (Choi et al., 2009). Interestingly, we found that infarcted hearts of STS-treated mice demonstrated a significantly reduced number of TUNEL-positive (apoptotic) cells in their border zones, as compared with vehicle-treated counterparts (Figure 4A). Results of additional immunodetection of cleaved caspase-3-positive apoptotic cells mirrored the results TUNEL assay indicating that STS treatment likely inhibited apoptosis. Also, the Western blot analysis of post-MI heart tissue demonstrated that levels of cleaved caspase-3 and Bax increased after MI were markedly attenuated after STS treatment (Figure 4C), while STS increased the level of Bcl-2 that was decreased in post-infarct hearts (Figure 4C).if To investigate the effect of STS on cardiomyocyte autophagy, we evaluated the presence of cellular structures associated with autophagy in post-MI hearts treated with STS via electron microscopy. Microscopy revealed an increase in both autophagic vacuoles and lysosomes in STS-treated hearts compared with those from vehicle-treated controls (Figure 5A). Also, the double immunofluorescent labeling with anti-LC3 and anti-myoglobin antibodies revealed that myocardium of the infarcted area of the vehicle-treated mice contained only few LC3-positive auto-phagocytic vacuoles (Figure 5B). In contrast, myocardium of STS-treated mice revealed significantly higher levels of LC3-positive immunofluorescence in surviving cardiomyocytes located on the border of the infarcted areas. Moreover, Western blot analysis of the tissues demonstrated that post-MI hearts of STS-treated mice displayed heightened ratio of LC3 lipidation (LC3II/LC3I) than the vehicle-treated controls, while the autophagy substrate p62 levels were However, this beneficial effect of STS disappeared when this compound was coadministered with the inhibitor of endocytosis, chloroquine (Figures 4B, C). FIGURE 4 \| STS attenuated apoptosis in post-infarct heart. (A) Representative immunofluorescent images and quantitative analysis of terminal deoxyuridine triphosphate nick end-labeling (TUNEL) staining in the heart from mice after left anterior descending artery ligation (scale bar = 40 µm, 4′,6-diamidino-2- phenylindole: blue, TUNEL: green). (B) Immunohistochemical analysis of caspase-3 (brown) expression in the infarct border zone of the heart and graphs showed the incidences of caspase-3 (scale bar = 40 µm). Sodium Tanshinone IIA Sulfonate Attenuated Apoptosis in Post-Infarct Hearts As previous research has shown that apoptosis plays a key role to maladaptive LV remodeling and dysfunction, we now FIGURE 3 \| STS attenuated myocardial remodeling in mice after MI. (A) Representative heart photographs and the ratio of heart weight to body weight (HW/BW) are shown (scale bar = 2 mm). (B, C) Histological preparations stained with Masson’s trichrome, showing the effects of treatment on scar circumference (scale bar = 5 mm) and ventricular fibrosis (scale bar = 40 µm). (D) Hematoxylin and eosin-stained LV sections (scale bar = 40 µm). Cross-sectional area of myocardium was calculated in 10 randomly selected fields from each LV section, and five sections for each heart were measured. (E) Western blot analysis for atrial natriuretic peptide (ANP) by use of an anti-ANP antibody. Expression of tubulin served as a loading control. Representative blots from three independent experiments are shown. (F) Real-time quantitative PCR analyses for the expression of ANP messenger RNA. 18S expression was used as the internal control. #P < 0.05 versus sham; P < 0.05 versus vehicle; &P < 0.05 versus STS. Veh, vehicle; STS, sodium tanshinone IIA sulfonate; Cq, chloroquine. FIGURE 3 \| STS attenuated myocardial remodeling in mice after MI. (A) Representative heart photographs and the ratio of heart weight to body weight (HW/BW) are shown (scale bar = 2 mm). (B, C) Histological preparations stained with Masson’s trichrome, showing the effects of treatment on scar circumference (scale bar = 5 mm) and ventricular fibrosis (scale bar = 40 µm). (D) Hematoxylin and eosin-stained LV sections (scale bar = 40 µm). Cross-sectional area of myocardium was calculated in 10 randomly selected fields from each LV section, and five sections for each heart were measured. (E) Western blot analysis for atrial natriuretic peptide (ANP) by use of an anti-ANP antibody. Expression of tubulin served as a loading control. Representative blots from three independent experiments are shown. (F) Real-time quantitative PCR analyses for the expression of ANP messenger RNA. 18S expression was used as the internal control. #P < 0.05 versus sham; P < 0.05 versus vehicle; &P < 0.05 versus STS. Veh, vehicle; STS, sodium tanshinone IIA sulfonate; Cq, chloroquine. July 2019 \| Volume 10 \| Article 779 5 Frontiers in Pharmacology \| www.frontiersin.org Mao et al. STS Attenuates Pathologic Cardiac Remodeling Sodium Tanshinone IIA Sulfonate Amplified Autophagy in the Post-Infarct Heartsf (B) Representative images of LC3 dots in ischemic border zone of post-MI heart; green, LC3; blue, 4′,6-diamidino-2- phenylindole-stained nuclei; red, myoglobin-positive cardiomyocytes; original magnification ×200 (scale bar = 20 μm). (C) Western blots with densitometric analysis (left panel) of autophagy-related LC3 and p62 protein expression in hearts. Graphs (right panel) show the intensity of each band in fold change: the LC3-II/LC3-I ratio and p62 expression. #P < 0.05 versus sham; P < 0.05 versus vehicle; &P < 0.05 versus STS. Veh, vehicle; STS, sodium tanshinone IIA sulfonate; Cq, chloroquine. FIGURE 5 \| Sustained activation of autophagy in hearts treated with STS. (A) Electron micrographs of autophagosomes in the ischemic border zone of heart myocardium 4 weeks after left anterior descending artery ligation. Original magnification ×5,000; (scale bars = 2 μm). Quantitative analysis of autophagosome number is shown in right panel. (B) Representative images of LC3 dots in ischemic border zone of post-MI heart; green, LC3; blue, 4′,6-diamidino-2- phenylindole-stained nuclei; red, myoglobin-positive cardiomyocytes; original magnification ×200 (scale bar = 20 μm). (C) Western blots with densitometric analysis (left panel) of autophagy-related LC3 and p62 protein expression in hearts. Graphs (right panel) show the intensity of each band in fold change: the LC3-II/LC3-I ratio and p62 expression. #P < 0.05 versus sham; P < 0.05 versus vehicle; &P < 0.05 versus STS. Veh, vehicle; STS, sodium tanshinone IIA sulfonate; Cq, chloroquine. Sodium Tanshinone IIA Sulfonate Inhibited Transforming Growth Factor β Signaling Pathway in Post-Infarct Hearts It has been previously demonstrated that activation of the transforming growth factor β1 (TGFβ1)/Smad pathway contributed to the healing and formation of the post-infarct myocardial scars in humans (Euler, 2015). On the other hand, recent evidence had also suggested that intracellular activation of sirtuin 1 (Sirt1) might contribute to limitation of the maladaptive remodeling of the post-infarct myocardium, by inhibiting the Frontiers in Pharmacology \| www.frontiersin.org Sodium Tanshinone IIA Sulfonate Amplified Autophagy in the Post-Infarct Heartsf (C) Western blot analysis of caspase-3, Bax, and Bcl-2 expression in post-infarct hearts. Expression of tubulin served as a loading control. Representative blots from three independent experiments are shown. #P < 0.05 versus sham; P < 0.05 versus vehicle; &P < 0.05 versus STS. Veh, vehicle; STS, sodium tanshinone IIA sulfonate; Cq, chloroquine. FIGURE 4 \| STS attenuated apoptosis in post-infarct heart. (A) Representative immunofluorescent images and quantitative analysis of terminal deoxyuridine triphosphate nick end-labeling (TUNEL) staining in the heart from mice after left anterior descending artery ligation (scale bar = 40 µm, 4′,6-diamidino-2- phenylindole: blue, TUNEL: green). (B) Immunohistochemical analysis of caspase-3 (brown) expression in the infarct border zone of the heart and graphs showed the incidences of caspase-3 (scale bar = 40 µm). (C) Western blot analysis of caspase-3, Bax, and Bcl-2 expression in post-infarct hearts. Expression of tubulin served as a loading control. Representative blots from three independent experiments are shown. #P < 0.05 versus sham; P < 0.05 versus vehicle; &P < 0.05 versus STS. Veh, vehicle; STS, sodium tanshinone IIA sulfonate; Cq, chloroquine. July 2019 \| Volume 10 \| Article 779 Frontiers in Pharmacology \| www.frontiersin.org 6 STS Attenuates Pathologic Cardiac Remodeling Mao et al. URE 5 \| Sustained activation of autophagy in hearts treated with STS. (A) Electron micrographs of autophagosomes in the ischemic border zone of heart cardium 4 weeks after left anterior descending artery ligation. Original magnification ×5,000; (scale bars = 2 μm). Quantitative analysis of autophagosome ber is shown in right panel. (B) Representative images of LC3 dots in ischemic border zone of post-MI heart; green, LC3; blue, 4′,6-diamidino-2- ylindole-stained nuclei; red, myoglobin-positive cardiomyocytes; original magnification ×200 (scale bar = 20 μm). (C) Western blots with densitometric ysis (left panel) of autophagy-related LC3 and p62 protein expression in hearts. Graphs (right panel) show the intensity of each band in fold change: the II/LC3-I ratio and p62 expression. #P < 0.05 versus sham; P < 0.05 versus vehicle; &P < 0.05 versus STS. Veh, vehicle; STS, sodium tanshinone IIA nate; Cq, chloroquine. FIGURE 5 \| Sustained activation of autophagy in hearts treated with STS. (A) Electron micrographs of autophagosomes in the ischemic border zone of heart myocardium 4 weeks after left anterior descending artery ligation. Original magnification ×5,000; (scale bars = 2 μm). Quantitative analysis of autophagosome number is shown in right panel. Sodium Tanshinone IIA Sulfonate Inhibited Transforming Growth Factor β Signaling Pathway in Post-Infarct Hearts respectively decreased (Figure 5C). We also noted that all above-mentioned molecular effects of STS were consistently reversed when coadministered with the potent blocker of endocytosis, chloroquine. It has been previously demonstrated that activation of the transforming growth factor β1 (TGFβ1)/Smad pathway contributed to the healing and formation of the post-infarct myocardial scars in humans (Euler, 2015). On the other hand, recent evidence had also suggested that intracellular activation of sirtuin 1 (Sirt1) might contribute to limitation of the maladaptive remodeling of the post-infarct myocardium, by inhibiting the Interestingly, LC3II/LC3I ratios and p62 expression were not affected by STS in sham-operated mice, suggesting STS could not induce autophagy in normal condition. In contrast, in the heart treated with the combination of STS plus chloroquine, the LC3-II/LC3-I ratios were similar to those seen in vehicle-treated hearts (Figure 5C). July 2019 \| Volume 10 \| Article 779 Frontiers in Pharmacology \| www.frontiersin.org 7 STS Attenuates Pathologic Cardiac Remodeling Mao et al. We found that levels of TGFβ1 in myocardium of MI mice, treated with STS, were significantly lower than those in heart tissues harvested from the vehicle-treated counterparts (Figures 6A, B). To further evaluate the role of the TGFβ signaling pathway in the STS-mediated cardioprotection, we also assessed the levels of its downstream components: Smad3 and Smad7 in post-infarct heart tissues. Interestingly, we found that STS treatment of MI mice was only able to downregulate levels of Smad3, while it actually caused an increase in Smad7 levels in myocardia from this experimental group (Figure 6A). Immunostaining of the parallel myocardial sections with the respective antibodies further suggested that treatment with STS indeed activated Sirt1 that subsequently caused inhibition of the TGF-β/Smad3 pathway (Figure 6B). TGFβ1/Smad3 pathway (Huang et al., 2014). As such, we sought to investigate the potential role of Sirt1 in the mechanistic pathway by which STS would attenuate the detrimental post-MI myocardial remodeling. Meaningfully, the obtained results indicated that treatment of MI mice with STS indeed leads to an increase in the expression of Sirt1 in their hearts (Figure 6A). Importantly, the Sirt1 level in the sham-operated mice was not affect by STS.h We found that levels of TGFβ1 in myocardium of MI mice, treated with STS, were significantly lower than those in heart tissues harvested from the vehicle-treated counterparts (Figures 6A, B). Sodium Tanshinone IIA Sulfonate Inhibited Transforming Growth Factor β Signaling Pathway in Post-Infarct Hearts To further evaluate the role of the TGFβ signaling pathway in the STS-mediated cardioprotection, we also assessed the levels of its downstream components: Smad3 and Smad7 in post-infarct heart tissues. Interestingly, we found that STS treatment of MI mice was only able to downregulate levels of Smad3, while it actually caused an increase in Smad7 levels in myocardia from this experimental group (Figure 6A). Immunostaining of the parallel myocardial sections with the respective antibodies further suggested that treatment with STS indeed activated Sirt1 that subsequently caused inhibition of the TGF-β/Smad3 pathway (Figure 6B). f Therefore, our novel data indicated that reported mechanism by which STS reduces pathologic cardiac remodeling may also involve the Sirt-1-triggered inhibition of the TGFβ1 signaling pathway. In order to further test this possibility, we used Western blot analysis and immunofluorescence to compare the levels of TGFβ1 in post-infarct hearts treated with either STS or a vehicle. FIGURE 6 \| Effects of STS on the TGFβ signaling pathway in post-infarct heart. (A) Representative Western blots and quantification of Sirt1/tubulin, TGFβ1/tubulin, Smad/tubulin, and Smad7/tubulin ratios in the hearts of sham-, vehicle-, or STS-treated mice. Horizontal lines display the mean ± SEM. (B) Representative images and color intensity analysis of immunohistochemistry of the infarct border zone after STS treatment (scale bar = 40 µm). #P < 0.05 versus sham; P < 0.05 versus vehicle; &P < 0.05 versus STS. Veh, vehicle; STS, sodium tanshinone IIA sulfonate; Cq, chloroquine. FIGURE 6 \| Effects of STS on the TGFβ signaling pathway in post-infarct heart. (A) Representative Western blots and quantification of Sirt1/tubulin, TGFβ1/tubulin, Smad/tubulin, and Smad7/tubulin ratios in the hearts of sham-, vehicle-, or STS-treated mice. Horizontal lines display the mean ± SEM. (B) Representative images and color intensity analysis of immunohistochemistry of the infarct border zone after STS treatment (scale bar = 40 µm). #P < 0.05 versus sham; P < 0.05 versus vehicle; &P < 0.05 versus STS. Veh, vehicle; STS, sodium tanshinone IIA sulfonate; Cq, chloroquine. FIGURE 6 \| Effects of STS on the TGFβ signaling pathway in post-infarct heart. (A) Representative Western blots and quantification of Sirt1/tubulin, TGFβ1/tubulin, Smad/tubulin, and Smad7/tubulin ratios in the hearts of sham-, vehicle-, or STS-treated mice. Horizontal lines display the mean ± SEM. (B) Representative images and color intensity analysis of immunohistochemistry of the infarct border zone after STS treatment (scale bar = 40 µm). Sodium Tanshinone IIA Sulfonate Inhibited Transforming Growth Factor β Signaling Pathway in Post-Infarct Hearts #P < 0.05 versus sham; P < 0.05 versus vehicle; &P < 0.05 versus STS. Veh, vehicle; STS, sodium tanshinone IIA sulfonate; Cq, chloroquine. July 2019 \| Volume 10 \| Article 779 Frontiers in Pharmacology \| www.frontiersin.org 8 Mao et al. STS Attenuates Pathologic Cardiac Remodeling showing both an increase in TIMP1 and a decrease in MMPs with STS administration post-MI (Figure 7B). Sodium Tanshinone IIA Sulfonate Altered the Matrix Metalloproteinases/ Metalloproteinases Balance in Post-Infarct Myocardium showing both an increase in TIMP1 and a decrease in MMPs with STS administration post-MI (Figure 7B). Sodium Tanshinone IIA Sulfonate Regulated of Anti-Adenosine Monophosphate Kinase/Mechanistic Target of Rapamycin/P70/S6K Pathway Results of previous experimental and clinical studies have already provided evidence for the existence of a cause–effect relationship between adverse alterations in the balance between matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) that may be critical for the myocardial matrix remodeling described in several cardiovascular conditions (Jacob, 2003). They also demonstrated that pharmacological inhibition of MMPs has become an enticing target for drug design in treating LV remodeling after MI (Cerisano et al., 2014). Therefore, we investigated whether the reported STS-mediated beneficial cardiac remodeling would also involve alteration expression or ultimate functions of selected MMPs. Results of our Western blot analysis demonstrated that treatment with STS leads to inhibition of the MMP2 and MMP9 expression while increasing the levels of TIMP1 (Figure 7A). Moreover, immunohistochemistry supported the molecular data Previous research has shown that the AMPK/mTOR signaling pathway plays an important role in cardioprotection against temporary ischemia or infarction (Lee et al., 2003; Yan et al., 2005). To investigate whether the STS-mediated improvement in remodeling would also involve alterations in this pathway, we assessed the expression and phosphorylation level of indicated proteins involved in this pathway. Results of our Western blot analysis revealed a significant increase in the levels of activated (phosphorylated) AMPK as well as a decrease in phosphorylated mTOR in the hearts of mice treated with STS (Figure 8A). In addition, levels of phosphorylated P70/S6K, a direct downstream target of mTOR, also decreased significantly FIGURE 7 \| Regulation of myocardial MMP/TIMP expression in STS treated post infarct heart (A) Western blots with densitometric analysis of levels MMP2 MMP9 FIGURE 7 \| Regulation of myocardial MMP/TIMP expression in STS-treated post-infarct heart. (A) Western blots with densitometric analysis of levels MMP2, MMP9, or TIMP1 (normalized to tubulin) in the hearts of sham-, vehicle-, or STS-treated mice. (B) Immunohistochemical images and color intensity analysis of MMP2, MMP9, and TIMP1 expression in the infarct border zone after STS treatment (scale bar = 40 µm). #P < 0.05 versus sham; P < 0.05 versus vehicle; &P < 0.05 versus STS. Veh, vehicle; STS, sodium tanshinone IIA sulfonate; Cq, chloroquine. FIGURE 7 \| Regulation of myocardial MMP/TIMP expression in STS-treated post-infarct heart. (A) Western blots with densitometric analysis of levels MMP2, MMP9, or TIMP1 (normalized to tubulin) in the hearts of sham-, vehicle-, or STS-treated mice. Sodium Tanshinone IIA Sulfonate Regulated of Anti-Adenosine Monophosphate Kinase/Mechanistic Target of Rapamycin/P70/S6K Pathway In contrast, protein levels of phospho-mTOR (Ser2448) and phospho-P70/S6K (Thr309) displayed a significant decrease after administration of STS following MI. (B) Immunohistochemical images and color intensity graphs of phosphorylated AMPK, mTOR, and P70/S6K in the infarct border zone after STS treatment (scale bar = 40 µm). #P < 0.05 versus sham; P < 0.05 versus vehicle. Veh, vehicle; STS, sodium tanshinone IIA sulfonate. FIGURE 8 \| STS regulates AMPK and mTOR activation in post-infarct hearts. (A) Western blot and densitometric analysis revealed a significant increase in the phosphorylation of AMPK (Thr172) after administration of STS. In contrast, protein levels of phospho-mTOR (Ser2448) and phospho-P70/S6K (Thr309) displayed a significant decrease after administration of STS following MI. (B) Immunohistochemical images and color intensity graphs of phosphorylated AMPK, mTOR, and P70/S6K in the infarct border zone after STS treatment (scale bar = 40 µm). #P < 0.05 versus sham; P < 0.05 versus vehicle. Veh, vehicle; STS, sodium tanshinone IIA sulfonate. Sodium Tanshinone IIA Sulfonate Regulated of Anti-Adenosine Monophosphate Kinase/Mechanistic Target of Rapamycin/P70/S6K Pathway (B) Immunohistochemical images and color intensity analysis of MMP2, MMP9, and TIMP1 expression in the infarct border zone after STS treatment (scale bar = 40 µm). #P < 0.05 versus sham; P < 0.05 versus vehicle; &P < 0.05 versus STS. Veh, vehicle; STS, sodium tanshinone IIA sulfonate; Cq, chloroquine. FIGURE 7 \| Regulation of myocardial MMP/TIMP expression in STS-treated post-infarct heart. (A) Western blots with densitometric analysis of levels MMP2, MMP9, or TIMP1 (normalized to tubulin) in the hearts of sham-, vehicle-, or STS-treated mice. (B) Immunohistochemical images and color intensity analysis of MMP2, MMP9, and TIMP1 expression in the infarct border zone after STS treatment (scale bar = 40 µm). #P < 0.05 versus sham; P < 0.05 versus vehicle; &P < 0.05 versus STS. Veh, vehicle; STS, sodium tanshinone IIA sulfonate; Cq, chloroquine. July 2019 \| Volume 10 \| Article 779 9 Frontiers in Pharmacology \| www.frontiersin.org STS Attenuates Pathologic Cardiac Remodeling Mao et al. STS-treated hearts, as compared with a vehicle-treated ntrols (Figure 8A) Effect of an AMPK Inhibitor on Sodium Tanshinone IIA Sulfonate-Induced IGURE 8 \| STS regulates AMPK and mTOR activation in post-infarct hearts. (A) Western blot and densitometric analysis revealed a significant increase in the hosphorylation of AMPK (Thr172) after administration of STS. In contrast, protein levels of phospho-mTOR (Ser2448) and phospho-P70/S6K (Thr309) displayed a gnificant decrease after administration of STS following MI. (B) Immunohistochemical images and color intensity graphs of phosphorylated AMPK, mTOR, and P70/S6 the infarct border zone after STS treatment (scale bar = 40 µm). #P < 0.05 versus sham; P < 0.05 versus vehicle. Veh, vehicle; STS, sodium tanshinone IIA sulfonate. FIGURE 8 \| STS regulates AMPK and mTOR activation in post-infarct hearts. (A) Western blot and densitometric analysis revealed a significant increase in the phosphorylation of AMPK (Thr172) after administration of STS. In contrast, protein levels of phospho-mTOR (Ser2448) and phospho-P70/S6K (Thr309) displayed a significant decrease after administration of STS following MI. (B) Immunohistochemical images and color intensity graphs of phosphorylated AMPK, mTOR, and P70/S6K in the infarct border zone after STS treatment (scale bar = 40 µm). #P < 0.05 versus sham; P < 0.05 versus vehicle. Veh, vehicle; STS, sodium tanshinone IIA sulfonate. FIGURE 8 \| STS regulates AMPK and mTOR activation in post-infarct hearts. (A) Western blot and densitometric analysis revealed a significant increase in the phosphorylation of AMPK (Thr172) after administration of STS. Frontiers in Pharmacology \| www.frontiersin.org Effect of an AMPK Inhibitor on Sodium Tanshinone IIA Sulfonate-Induced Anti-Remodeling in STS-treated hearts, as compared with a vehicle-treated controls (Figure 8A). Importantly, results of the parallel immunohistochemistry further confirmed our Western blot analysis, showing increased levels of phosphorylated AMPK and decreased levels of phosphorylated mTOR and P70/S6K in the post-MI hearts treated with STS (Figure 8B). Given that STS was able to stimulate AMPK phosphorylation, we further tested the putative dependence of STS signaling on AMPK function. Therefore, in the next set of experiments, the STS was administered to post-infarct mice in the presence and absence of compound C (a specific pharmacological AMPK inhibitor). We found that myocardia of post-MI mice, simultaneously treated with compound C and STS, demonstrated At this point, we have to also mention that in the sham- operated mice, STS treatment did not induce any upregulation in the AMPK/mTOR signaling pathway. July 2019 \| Volume 10 \| Article 779 Frontiers in Pharmacology \| www.frontiersin.org 10 STS Attenuates Pathologic Cardiac Remodeling Mao et al. to its role in opening of mitochondrial ATP channels (Khan et al., 2006). On the other hand, results derived from a Langendorff model of ischemia/reperfusion published by Kis and colleagues showed that inhibition of mTOR before onset of ischemia lessened the cardioprotective effect of ischemic precondition (Kis et al., 2003). Despite the fact that beneficial effects of rapamycin in the ischemia/reperfusion model have been a topic of contention, we have also addressed this problem in the present study and were able to demonstrate that the cardioprotective effect of STS was associated with induction of rapamycin-dependent inhibition of mTOR pathway in our experimental model of MI. Moreover, we also tested whether the observed cardioprotective effect of STS treatment would also depend on elimination of yet another main downstream targets of mTOR pathway, P70/S6K, which enhanced synthesis has been previously linked to deleterious cardiac hypertrophy that could be prevented by rapamycin pretreatment in several animal models of MI (McMullen et al., 2004b). Meaningfully, we found that synthesis of P70/S6K protein was significantly upregulated in the hearts of infarcted mice (as compared with sham-operated controls) and that treatment with STS that induced their clinically observed cardioprotection also associated with reduction of their infarct-induced elevation of P70/S6K protein expression to the levels detected in sham- operated controls. Importantly, these results corroborated well with our findings that STS stimulated AMPK activity and autophagy while suppressing the activation of mTOR and P70/ S6K. DISCUSSION The main finding of this study was that treatment with STS partially reversed the progression of adverse LV remodeling following MI and did not induce any side effects. Our results suggested that the beneficial actions of STS, in a large part, depend on its ability of the potent stimulation of autophagy of dying hypoxic cardiomyocytes and endocytic removal of the fragmented extracellular matrix, which persisted presence in the injured , but untreated myocardium, causes a local release of numerous growth factors and cytokines that consequently complicate the normal healing of the infarcted myocardium and stimulate pathological cardiac remodeling (Wu et al., 2017). These data were consistent with other reports where increased autophagy led to improved cardiac performance and cardiomyocytes survival in the ischemic heart (Matsui et al., 2007; Kanamori et al., 2009). Moreover, our assumption was further endorsed by the fact that additional treatment of MI mice with chloroquine, a potent inhibitor of cytoskeleton assembly that consequently eliminates the endocytosis and intracellular transport, completely eradicated the beneficial effects of STS treatment. g After establishing that treatment of MI mice with STS consistently induced the inhibition of the maladaptive remodeling of their myocardium, that we mechanistically linked to triggering of the AMPK/mTOR/P70/S6K signaling pathway, we also tested whether STS could be also involved in modulation of other key molecules, including nicotine adenine dinucleotide-dependent protein/histone deacetylase (Sirt1) and TGFβ1 that have been previously linked to cardioprotection. We particularly turned attention to Sirt1, in which overexpression was previously linked to a reduction of the ischemia/reperfusion-induced cardiac injury, through stimulation of autophagy of the ischemic cardiomyocytes and to amelioration of cardiac fibrosis by inhibition of the downstream TGFβ1/Smad3 signaling pathway (Huang et al., 2014; Hao et al., 2016). Importantly, our results, presented in the present, clearly demonstrated beneficial effects of the STS treatment, detected in infarcted murine hearts, which was associated with an activation of Sirt1 and a consequent suppression of TGF-β1 and Smad3 levels. The next part of our study was inspired by the previously reported observations that indicated that all active movement and recycling of all cellular vesicles involved in the autophagy is highly energy dependent and that a major integrator of the metabolic response to changes in energy availability, the AMP kinase, is activated by ATP depletion and causes accumulation of AMP (Inoki et al., 2003; Kanamori et al., 2011). Effect of an AMPK Inhibitor on Sodium Tanshinone IIA Sulfonate-Induced Anti-Remodeling These reports are consistent with our findings that STS stimulated AMPK activity and autophagy while suppressing the activation of mTOR and P70/S6K (Chung et al., 1992). remarkably lower levels of phosphorylated AMPK, while their mTOR levels did not differ from levels seen in vehicle-treated controls (Figure 9A). Importantly, we have established that inhibition of AMPK signaling practically abolished the beneficial effects of STS on cardiac structure and function, including elevation of LV internal dimension at end diastole, decreased cardiomyocyte hypertrophy, lower myocardial fibrosis, and decrease of LV ejection fraction or FS (Figure 9B). Looking at its role in autophagy, coadministration of STS with compound C eliminated the increase in autophagy seen in mice treated with STS alone, an effect seen in both Western blot and immunofluorescence (Figure 9B). Taken together, all of the above results suggest that AMPK-mediated autophagy plays an essential role in STS-induced cardiac protection against post-MI remodeling. Frontiers in Pharmacology \| www.frontiersin.org DISCUSSION These data strongly suggested that treatment with STS also contributes to the post-MI remodeling by modulating the optimal balance of the ECM turnover, in the final healing phase of this lifesaving process. AUTHOR CONTRIBUTIONS SM drafted this manuscript; SM, MZ, and MV performed the experiments; MC made statistical analysis; AH made critical revision of the manuscript and contributed to the rationalization of the study. All authors read and approved the final manuscript. ETHICS STATEMENT This study was carried out in accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals published by the US National Academy of Sciences (8th edition, Washington DC, 2011). The protocol was approved by the Institutional Animal Care and Use Committee of Guangdong Province Hospital of Chinese Medicine, Guangzhou University of Traditional Chinese Medicine. DISCUSSION However, the increase in phosphorylated AMPK, observed in STS-treated hearts, was accompanied by an increase in the levels of ATP, a cellular environment that practically would not favor further activation of AMPK. This suggests that STS activated AMP kinase, likely via a different pathway than the AMP-mediated activation of this important enzyme (Ikeda et al., 2009). It has been previously well documented that abnormalities in synthesis and degradation of cardiac extracellular matrix (ECM) after MI may eventually lead to the loss of the resilient elastic fibers and to an excessive accumulation of fibrotic collagens that contributes to formation of the rigid scars, mechanical stiffness of the myocardium, and the subsequent impairment of the cardiac function. The main proteases responsible for degradation of practically all ECM proteins Indeed, it has been previously demonstrated that AMP kinase serves as a positive regulator of autophagy mainly via inhibition of the mTOR complex-dependent pathway and that application of rapamycin causes inhibition of LV remodeling after MI (McMullen et al., 2004a; Shaw, 2009). The rapamycin- dependent reduction of the infarct size has been also linked July 2019 \| Volume 10 \| Article 779 Frontiers in Pharmacology \| www.frontiersin.org 11 Mao et al. STS Attenuates Pathologic Cardiac Remodeling ued ed FIGURE 9 \| Continued July 2019 \| Volume 10 \| Article 779 Frontiers in Pharmacology \| www.frontiersin.org 12 STS Attenuates Pathologic Cardiac Remodeling Mao et al. FIGURE 9 \| Effects of AMPK inhibition on STS-induced cardiac protection. (A) Western blots with densitometric analysis of phospho-AMPK (Thr172) and phospho- mTOR (Ser2448). Graphs show the intensity of each band in arbitrary units. (B) Representative pictures of echocardiography, Masson’s trichrome-stained LV micrographs, hematoxylin and eosin-stained LV micrographs, electron micrographs showing autophagic vacuoles, and immunofluorescent micrographs of LC3 in ischemic border zone of heart. Compound C suppressed STS-induced autophagy along with anti-remodeling effects. #P < 0.05 versus sham; *P < 0.05 versus vehicle; &P < 0.05 versus STS. Veh, vehicle; STS, sodium tanshinone IIA sulfonate; CC, compound C. belong to the family of MMPs. In particular, multifunctional proteinases (MMP9 and MMP2) capable to degrade both major ECM components, collagen and elastin, have been implicated in the progression of myocardium ischemic lesions that contribute to the expansion of the infarct zone. Meaningfully, the targeted deletion of MMP2 or MMP9 reduced the rate of post-MI cardiac rupture and provided partial protection against LV dilation and dysfunction. CLINICAL IMPLICATIONS Despite the diverse treatment strategies constantly improving the adverse LV remodeling after MI, a substantial proportion of patients afflicted by MI still develop the detrimental complications. The presented results of our experimental study provided the substantial mechanistic explanation how STS would attenuate the deleterious myocardial remodeling of the MI in the animal model. Thus, the obtained results not only provide a new insight for our understanding of the MI pathology but also justify the use of this compound, derived from the traditional Chinese medicine as an effective factor supporting the strict pharmacological regimen recommended for the clinical treatments of MI patients. g It has been reported that heightened apoptosis of cardiomyocytes and their consequent death mechanistically contribute to the progression of post-MI LV remodeling and dysfunction (Kanamori et al., 2007). In addition, it has been also suggested that the activation of Sirt1 protects cardiac cells against apoptosis by an autophagy-dependent pathway (Ben Salem et al., 2017). Consistently, we also noticed that myocardia of the STS-treated mice demonstrated the evident absence of the immune-detected LC3 antigen and a significantly fewer TUNEL-immuno-positive cells. This was in contrast to myocardial samples derived from the untreated control MI mice that contained clusters of evidently TUNEL-positive cells in the infarcted regions. Thus, we conclude that treatment with STS also augmented the apoptosis of hypoxic, but still alive cardiomyocytes in the infarcted regions. It has been reported that heightened apoptosis of cardiomyocytes and their consequent death mechanistically contribute to the progression of post-MI LV remodeling and dysfunction (Kanamori et al., 2007). In addition, it has been also suggested that the activation of Sirt1 protects cardiac cells against apoptosis by an autophagy-dependent pathway (Ben Salem et al., 2017). Consistently, we also noticed that myocardia of the STS-treated mice demonstrated the evident absence of the immune-detected LC3 antigen and a significantly fewer TUNEL-immuno-positive cells. This was in contrast to myocardial samples derived from the untreated control MI mice that contained clusters of evidently TUNEL-positive cells in the infarcted regions. Thus, we conclude that treatment with STS also augmented the apoptosis of hypoxic, but still alive cardiomyocytes in the infarcted regions. STUDY LIMITATIONS In this study, we have found that STS exerts a beneficial effect on post-MI remodeling and have determined that this effect is the result of stimulation of cellular autophagy via AMPK activation through experiments involving chloroquine and compound C. Although we demonstrated that neither of these pharmacological agents produced significant side effects DISCUSSION On the other hand, it has also been reported that the experimentally induced deficiency of natural MMP’s inhibitor (TIMP1) in transgenic mice significantly exacerbated their LV remodeling after experimental MI, as compared with the MI-injured wild-type mice. Therefore, we also decided to test whether the treatment with STS would possibly modulate the balance between expressions of MMPs engaged in extracellular matrix remodeling and their natural inhibitor TIMP1, in myocardium of MI-injured mice (Jo et al., 2011). Importantly, the obtained results indicate that treatment of experimental mice with STS not only diminished levels of MMP2 and MMP9 in their infarcted myocardium but also, at the same time, upregulated expression of TIMP1. These data strongly suggested that treatment with STS also contributes to the post-MI remodeling by modulating the optimal balance of the ECM turnover, in the final healing phase of this lifesaving process. in mice, this approach has limitations in its specificity. To resolve this issue, we plan to further study the role of AMPK and autophagy in the actions of STS using perhaps knockout mice or small interfering RNA technology to more specifically inhibit these proteins and definitively determine the pathway of STS action. belong to the family of MMPs. In particular, multifunctional proteinases (MMP9 and MMP2) capable to degrade both major ECM components, collagen and elastin, have been implicated in the progression of myocardium ischemic lesions that contribute to the expansion of the infarct zone. Meaningfully, the targeted deletion of MMP2 or MMP9 reduced the rate of post-MI cardiac rupture and provided partial protection against LV dilation and dysfunction. On the other hand, it has also been reported that the experimentally induced deficiency of natural MMP’s inhibitor (TIMP1) in transgenic mice significantly exacerbated their LV remodeling after experimental MI, as compared with the MI-injured wild-type mice. Therefore, we also decided to test whether the treatment with STS would possibly modulate the balance between expressions of MMPs engaged in extracellular matrix remodeling and their natural inhibitor TIMP1, in myocardium of MI-injured mice (Jo et al., 2011). Importantly, the obtained results indicate that treatment of experimental mice with STS not only diminished levels of MMP2 and MMP9 in their infarcted myocardium but also, at the same time, upregulated expression of TIMP1. REFERENCES Inoki, K., Zhu, T., and Guan, K. L. (2003). TSC2 mediates cellular energy response to control cell growth and survival. Cell 115, 577–590. doi: 10.1016/ S0092-8674(03)00929-2 Ben Salem, I., Boussabbeh, M., Da Silva, J. 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Good and bad sides of TGFbeta-signaling in myocardial infarction. Front. Physiol. 6, 66. doi: 10.3389/fphys.2015.00066 French, J. K., Hellkamp, A. S., Armstrong, P. W., Cohen, E., Kleiman, N. S., O’connor, C. M., et al. (2010). Mechanical complications after percutaneous coronary intervention in ST-elevation myocardial infarction (from APEX- AMI). Am. J. Cardiol. 105, 59–63. doi: 10.1016/j.amjcard.2009.08.653 Lee, M., Hwang, J. T., Lee, H. J., Jung, S. N., Kang, I., Chi, S. G., et al. (2003). AMP-activated protein kinase activity is critical for hypoxia-inducible factor-1 transcriptional activity and its target gene expression under hypoxic conditions in DU145 cells. J. Biol. Chem. 278, 39653–39661. doi: 10.1074/jbc.M306104200 Guo, R., Hu, N., Kandadi, M. R., and Ren, J. (2012). Facilitated ethanol metabolism promotes cardiomyocyte contractile dysfunction through autophagy in murine hearts. Autophagy 8, 593–608. doi: 10.4161/auto.18997 Long, R., You, Y., Li, W., Jin, N., Huang, S., Li, T., et al. (2015). Sodium tanshinone IIA sulfonate ameliorates experimental coronary no-reflow phenomenon through down-regulation of FGL2. Life Sci. 142, 8–18. doi: 10.1016/j.lfs.2015.10.018 Hao, Y., Lu, Q., Yang, G., and Ma, A. (2016). Lin28a protects against postinfarction myocardial remodeling and dysfunction through Sirt1 activation and autophagy enhancement. Biochem. Biophys. Res. Commun. 479, 833–840. doi: 10.1016/j.bbrc.2016.09.122 Mao, S., Chen, P. P., Li, T., Guo, L. H., and Zhang, M. Z. (2018). FUNDING This work was financially supported by the National Science Foundation (nos. 81703877, 81703848, and 81673702), Guangdong Province Pearl River Scholar Funded Scheme July 2019 \| Volume 10 \| Article 779 Frontiers in Pharmacology \| www.frontiersin.org 13 Mao et al. STS Attenuates Pathologic Cardiac Remodeling ACKNOWLEDGMENTS (2019 to SM), the National Basic Research Program of China (973 Program, 2015CB554400), the Science Foundation of Guangdong Province (nos. 2016A030313636, 2017A030310123, and 2017A030313725), the Medical Science and Technology Research Foundation of Guangdong Province (A2016192), the Department of Science and Technology of Guangdong Province (no. 2014A020221044), and Guangdong Provincial Hospital of Chinese Medicine program (YN2015QN15). The sponsors have had no role in the project development, in the collection of data, in the preparation of this manuscript, nor the decision to publish. The authors gratefully acknowledge the contributions of all the investigators of the program. SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fphar.2019.00779/ full#supplementary-material REFERENCES Tongguan capsule mitigates post-myocardial infarction remodeling by promoting autophagy and inhibiting apoptosis: role of Sirt1. Front. Physiol. 9, 15. doi: 10.3389/ fphys.2018.00589 Hu, Q., Wei, B., Wei, L., Hua, K., Yu, X., Li, H., et al. (2015). Sodium tanshinone IIA sulfonate ameliorates ischemia-induced myocardial inflammation and lipid accumulation in Beagle dogs through NLRP3 inflammasome. Int. J. Cardiol. 196, 183–192. doi: 10.1016/j.ijcard.2015.05.152 Mao, S., Li, X., Wang, L., Yang, P. C., and Zhang, M. (2015a). Rationale and design of sodium tanshinone IIA sulfonate in left ventricular remodeling secondary to acute myocardial infarction (STAMP-REMODELING) trial: a randomized controlled study. Cardiovasc. Drugs Ther. 29, 535–542. doi: 10.1007/s10557-015-6625-2 Huang, X. Z., Wen, D., Zhang, M., Xie, Q., Ma, L., Guan, Y., et al. (2014). Sirt1 activation ameliorates renal fibrosis by inhibiting the TGF-beta/Smad3 pathway. J. Cell. Biochem. 115, 996–1005. doi: 10.1002/jcb.24748 h Mao, S., Wang, L., Zhao, X., Shang, H., Zhang, M., and Hinek, A. (2015b). Sodium tanshinone IIA sulfonate for reduction of periprocedural myocardial injury during percutaneous coronary intervention (STAMP trial): rationale and design. Int. J. Cardiol. 182, 329–333. doi: 10.1016/j.ijcard.2014.12.166 Ikeda, Y., Sato, K., Pimentel, D. R., Sam, F., Shaw, R. J., Dyck, J. R., et al. (2009). Cardiac-specific deletion of LKB1 leads to hypertrophy and dysfunction. J. Biol. Chem. 284, 35839–35849. doi: 10.1074/jbc.M109.057273 July 2019 \| Volume 10 \| Article 779 Frontiers in Pharmacology \| www.frontiersin.org 14 Mao et al. STS Attenuates Pathologic Cardiac Remodeling cardioprotective effect of sodium tanshinone IIA sulphonate in isoproterenol- induced myocardial infarction in rats. Atherosclerosis 230, 148–156. doi: 10.1016/j.atherosclerosis.2013.07.005 Mao, S., Wang, Y., Zhang, M., and Hinek, A. (2014). Phytoestrogen, tanshinone IIA diminishes collagen deposition and stimulates new elastogenesis in cultures of human cardiac fibroblasts. Exp. Cell. Res. 323, 189–197. doi: 10.1016/j. yexcr.2014.02.001 Wu, P., Yuan, X., Li, F., Zhang, J., Zhu, W., Wei, M., et al. (2017). Myocardial upregulation of cathepsin D by ischemic heart disease promotes autophagic flux and protects against cardiac remodeling and heart failure. Circ. Heart Fail. 10, e004044. doi: 10.1161/CIRCHEARTFAILURE.117.004044 Matsui, Y., Takagi, H., Qu, X., Abdellatif, M., Sakoda, H., Asano, T., et al. (2007). Distinct roles of autophagy in the heart during ischemia and reperfusion: roles of AMP-activated protein kinase and Beclin 1 in mediating autophagy. Circ. Res. 100, 914–922. doi: 10.1161/01.RES.0000261924.76669.36 Wu, W. Y., Wang, W. Y., Ma, Y. L., Yan, H., Wang, X. B., Qin, Y. L., et al. (2013). 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Deletion of ribosomal S6 kinases does not attenuate pathological, physiological, or insulin-like growth factor 1 receptor- phosphoinositide 3-kinase-induced cardiac hypertrophy. Mol. Cell. Biol. 24, 6231–6240. doi: 10.1128/MCB.24.14.6231-6240.2004 Yang, R., Liu, A., Ma, X., Li, L., Su, D., and Liu, J. (2008). Sodium tanshinone IIA sulfonate protects cardiomyocytes against oxidative stress-mediated apoptosis through inhibiting JNK activation. J. Cardiovasc. Pharmacol. 51, 396–401. doi: 10.1097/FJC.0b013e3181671439 Qi, J., Yu, J., Tan, Y., Chen, R., Xu, W., Chen, Y., et al. (2017). Mechanisms of Chinese Medicine Xinmailong’s protection against heart failure in pressure-overloaded mice and cultured cardiomyocytes. Sci. Rep. 7, 42843. doi: 10.1038/srep42843 Zhou, G. Y., Zhao, B. L., Hou, J. W., Ma, G. E., and Xin, W. J. (1999). Protective effects of sodium tanshinone IIA sulphonate against adriamycin-induced lipid peroxidation in mice hearts in vivo and in vitro. Pharmacol. Res. 40, 487–491. doi: 10.1006/phrs.1999.0545 Qiu, X., Miles, A., Jiang, X., Sun, X., and Yang, N. (2012). Sulfotanshinone sodium injection for unstable angina pectoris: a systematic review of randomized controlled trials. Evid. Based Complement. Alternat. Med. 2012, 715790. doi: 10.1155/2012/715790 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. 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. Shaw, R. J. (2009). LKB1 and AMP-activated protein kinase control of mTOR signalling and growth. Acta. Physiol. (Oxf.) 196, 65–80. doi: 10.1111/j.1748-1716.2009.01972.x Copyright © 2019 Mao, Vincent, Chen, Zhang and Hinek. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). July 2019 \| Volume 10 \| Article 779 Frontiers in Pharmacology \| www.frontiersin.org 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. Springeling, T., Kirschbaum, S. W., Rossi, A., Baks, T., Karamermer, Y., Schulz, C., et al. (2013). Late cardiac remodeling after primary percutaneous coronary intervention-five-year cardiac magnetic resonance imaging follow-up. Circ. J. 77, 81–88. doi: 10.1253/circj.CJ-12-0043 Wei, B., You, M. G., Ling, J. J., Wei, L. L., Wang, K., Li, W. W., et al. (2013). Regulation of antioxidant system, lipids and fatty acid beta-oxidation contributes to the July 2019 \| Volume 10 \| Article 779 Frontiers in Pharmacology \| www.frontiersin.org 15
https://openalex.org/W3109719154	https://cronfa.swan.ac.uk/Record/cronfa55848/Download/55848__18839__b5070ab0e48140b3bfb33592a007fdde.pdf	English	null	3D Bioprinting and the Future of Surgery	Frontiers in surgery	2,020	cc-by	8,724	3D Bioprinting and the Future of Surgery Thomas H. Jovic 1,2, Emman J. Combellack 1,2, Zita M. Jessop 1,2 and Iain S. Whitaker 1,2* 1 Reconstructive Surgery and Regenerative Medicine Research Group, Swansea University, Swansea, United Kingdom, 2 Welsh Centre for Burns and Plastic Surgery, Morriston Hospital, Swansea, United Kingdom Thomas H. Jovic 1,2, Emman J. Combellack 1,2, Zita M. Jessop 1,2 and Iain S. Whitaker 1,2* 1 Reconstructive Surgery and Regenerative Medicine Research Group, Swansea University, Swansea, United Kingdom, 2 Welsh Centre for Burns and Plastic Surgery, Morriston Hospital, Swansea, United Kingdom Introduction: The disciplines of 3D bioprinting and surgery have witnessed incremental transformations over the last century. 3D bioprinting is a convergence of biology and engineering technologies, mirroring the clinical need to produce viable biological tissue through advancements in printing, regenerative medicine and materials science. To outline the current and future challenges of 3D bioprinting technology in surgery. Methods: A comprehensive literature search was undertaken using the MEDLINE, EMBASE and Google Scholar databases between 2000 and 2019. A narrative synthesis of the resulting literature was produced to discuss 3D bioprinting, current and future challenges, the role in personalized medicine and transplantation surgery and the global 3D bioprinting market. Edited by: Warren Matthew Rozen, Monash University, Australia Reviewed by: Vijayavenkataraman Sanjairaj, New York University Abu Dhabi, United Arab Emirates Michael P. Chae, Peninsula Health, Australia Rachna Ram, Monash Health, Australia Reviewed by: Vijayavenkataraman Sanjairaj, New York University Abu Dhabi, United Arab Emirates Michael P. Chae, Peninsula Health, Australia Rachna Ram, Monash Health, Australia Conclusion: Effective translation requires the input of scientists, engineers, clinicians, and regulatory bodies: there is a need for a collaborative effort to translate this impactful technology into a real-world healthcare setting and potentially transform the future of surgery. Correspondence: Iain S. Whitaker iainwhitaker@fastmail.fm orcid.org/0000-0002-3922-2079 Specialty section: This article was submitted to Reconstructive and Plastic Surgery, a section of the journal Frontiers in Surgery Keywords: 3D printing, transplantation, biotechnology, bioprinting, reconstruction INTRODUCTION Received: 24 September 2020 Accepted: 06 November 2020 Published: 27 November 2020 The advent of three-dimensional (3D) printing has evoked a global industrial revolution, garnering the attention of the public and media in the process. Despite having its roots in the automotive, packaging and architectural domains (1), major developments in 3D printing technology have born witness to an expanded role of printing technologies, spanning into healthcare research and prompting the development of numerous medical devices, models and prosthetics. REVIEW published: 27 November 2020 doi: 10.3389/fsurg.2020.609836 3D Bioprinting and the Future of Surgery Thomas H. Jovic 1,2, Emman J. Combellack 1,2, Zita M. Jessop 1,2 and Iain S. Whitaker 1,2 1 Reconstructive Surgery and Regenerative Medicine Research Group, Swansea University, Swansea, United Kingdom, 2 Welsh Centre for Burns and Plastic Surgery, Morriston Hospital, Swansea, United Kingdom Keywords: 3D printing, transplantation, biotechnology, bioprinting, reconstruction Results: The next 20 years will see the advent of bioprinted implants for surgical use, however the path to clinical incorporation will be fraught with an array of ethical, regulatory and technical challenges of which each must be surmounted. Previous clinical cases where regulatory processes have been bypassed have led to poor outcomes and controversy. Speculated roles of 3D bioprinting in surgery include the production of de novo organs for transplantation and use of autologous cellular material for personalized medicine. The promise of these technologies has sparked an industrial revolution, leading to an exponential growth of the 3D bioprinting market worth billions of dollars. Edited by: Warren Matthew Rozen, Monash University, Australia Citation: Diﬀerences in patient’s genotypes will aﬀect processes such as cell migration, post-printing phenotype, oncogenic potential (particularly in immortalized cell populations) and dysregulated diﬀerentiation, such as fat derived stem cells producing ectopic bone for example (14, 15). Teratoma formation and the recurrence or potentiation of malignancy from the use of stem cells remains a signiﬁcant scientiﬁc concern: a ﬁrst in human trial of induced pluripotent stem cells in Japan was ceased due to the genomic mutations that developed (16, 17). p 3D printing holds incredible potential for the future of surgery, as acknowledged by the Royal College of Surgeons in the Commission on the Future of Surgery (4). The biological applications of 3D printing technology, or “bioprinting,” traverse the disciplines of human biology, materials science and mechanical engineering, and incorporates this into clinical practice to yield novel and personalized surgical options for patients (5, 6). Successful implementation could lead to a paradigm shift in surgical outcomes, with the potential to obviate the need for donor organs for transplantation surgery and oﬀering the restoration of form and function without painful and destructive donor sites (7). Throughout the course of this review article we aim to identify the key roles this technology may play in the future of surgery and explore the pivotal considerations and challenges that remain to be addressed prior to the integration of three-dimensional printing and bioprinting into mainstream surgical practice. Challenges of Biological Ink (Bioink) Selection Irrespective of cell sources, the materials selected for bioink production must be biocompatible before being considered for use in humans. The immunogenicity and toxicity of bioinks will necessitate further investigation prior to human trials (18). Many materials are derived from non-human organisms such as alginate from seaweed and gelatin from porcine material. The foreign nature of these components risk immunogenicity, inﬂammation and infection (12, 19). The production of degradable biomaterials has attracted signiﬁcant research interest due to their potential to create scaﬀolds that resorb, as new tissue forms to replace it (18). Further research is also needed to investigate the risk of toxicity as by-products are released into bloodstream, and undergo renal or hepatic clearance (20). The bioprinting process itself, in particular for extrusion based bioprinting, may exert shear forces on cells. Mechanotransduction pathways may disrupt cell behavior and direct stem cells down certain, potentially undesired lineages (21). Citation: Jovic TH, Combellack EJ, Jessop ZM and Whitaker IS (2020) 3D Bioprinting and the Future of Surgery. Front. Surg. 7:609836. doi: 10.3389/fsurg.2020.609836 Surgery too has witnessed incremental transformations over the past century, with the introduction of microsurgery, transplantation and robotics augmenting the array of treatments available for patients. As the scope and complexity of surgical interventions broadens so too does November 2020 \| Volume 7 \| Article 609836 Frontiers in Surgery \| www.frontiersin.org 1 3D Bioprinting: Future of Surgery Jovic et al. the need to adequately plan and prepare for surgery. Furthermore, many procedures, particularly in reconstructive and transplantation surgery remain hindered by the availability of donor tissues and organs, the morbidity associated with tissue harvest and the potential complications related to immunosuppression (2, 3). 3D printing software can be used to extract digital data from patient images such as computed tomography, magnetic resonance imaging or laser scanning to yield custom-made and personalized constructs for surgical planning and implantation (1). In particular, the incorporation of a biological component would transform this established technology, with the potential to revolutionize personalized healthcare through the advent of autologous living implants akin to the patient’s own tissue. Challenges of Cell Source Selection Within the healthcare setting itself, the origins of both cell sources and bioink materials may spark further debate. Firstly, cells used to create simple tissue structures such as heart valves could feasibly be derived from either animals or humans, as with the porcine valves currently used in clinical practice. Animal sources are likely to enable greater mass production of tissue for surgical use but consist of allogenic material with a risk of disease xenotransmission (12). In contrast, human sources oﬀer greater biocompatibility and the opportunity for personalisation, but their use is likely to be fraught with tighter regulation, lengthier production times and higher costs. Many donor related ethical concerns could be bypassed by the use of autologous cell sources, however the accessibility of certain cell types and the presence of genetic diseases may cloud the ethical and regulatory aspects of autologous cell sourcing (13). In addition, due to limited human trials of successful clinical translation of tissue engineered constructs at present, there remains an element of unpredictability regarding how autologous cells will behave. The biological component of implants make integration and interactions more unpredictable when inserted into hosts than currently used stents, pacemakers and artiﬁcial joints. Citation: Furthermore, many hydrogel materials used as bioinks are required to be crosslinked post-printing to maintain their 3D shape. Crosslinking often requires chemical, thermal or enzymatic catalysis which may be cytotoxic or induce DNA damage, an example of which is the use of ultraviolet light as a photopolymerisation agent (22). For many of these crosslinking processes, the genotoxic eﬀects of free radicals and The Promise of 3D Bioprinting The introduction of 3D printing into surgical practice is already underway. An example of successful integration is the ability to 3D print customized titanium prosthetics which has revolutionized personalized maxillofacial surgery in the UK (8), and the ability to emulate the success of this technology in 3D printing tissue holds the potential to revolutionize transplantation surgery and reconstructive surgery (9). It is the promise of a biological component in technologies such as bioprinting that presents the most signiﬁcant challenges to 3D printing in surgery. There have been a number of recent reports that have raised concerns regarding the adoption of regenerative medical interventions such as 3D bioprinting into mainstream clinical practice (10). The process of bioprinting requires cells, bioinks and bioprinters, each of which presents biological, technical and ethical challenges and uncertainty regarding clinical eﬀectiveness and cost- eﬀectiveness (11). As such, the translation of 3D bioprinting into mainstream clinical practice will be fraught with signiﬁcant challenges (Figure 1). November 2020 \| Volume 7 \| Article 609836 Frontiers in Surgery \| www.frontiersin.org 2 Jovic et al. 3D Bioprinting: Future of Surgery FIGURE 1 \| Challenges for clinical translation in 3D bioprinting. The main challenges to clinically translating bioprinting technology traverse in vitro, in vivo and clinical domains, requiring the support of ﬁnancial investment, a robust logistical network and engagement from multidisciplinary professionals. FIGURE 1 \| Challenges for clinical translation in 3D bioprinting. The main challenges to clinically translating bioprinting technology traverse in vitro, in vivo and clinical domains, requiring the support of ﬁnancial investment, a robust logistical network and engagement from multidisciplinary professionals. ultraviolet irradiation on DNA damage may not be immediately apparent (22). cost eﬀectiveness and biography (11). The classiﬁcation of complex and novel regenerative and gene medicinal products was expanded to include tissue engineered constructs which sought to deﬁne Advanced therapy medicinal products (ATMPs) (23). This class of innovative therapies represents a novel group of therapeutic agents with signiﬁcant diﬀerences to those therapies currently licensed and available on the market (24). In an eﬀort to standardize market availability within the European Union (EU) the European Commission (EC) established the ATMP Regulation (EC 1394/2007) alongside directive 2009/120 which created deﬁnitions for these novel technologies alongside marketing authorization (MA) guidance (25). The Promise of 3D Bioprinting As one of the four product types covered by the ATMP umbrella, Tissue engineered products (TEPs) have seen a slow progression over the last 10 years, with relatively few transitioning from concept to patient application. The complexity of UK and EU regulation, coupled with challenges at a regional level with safety, scalability Frontiers in Surgery \| www.frontiersin.org Ethical Challenges The design of clinical trials will also prove challenging: it would be unethical to trial tissue engineered organ transplantation on healthy volunteers, and the use of patient speciﬁc cell populations mean that the patient themselves would need to act as their own control, introducing a high degree of heterogeneity when attempting to assess treatment eﬃcacy (13). This could be particularly problematic when interpreting favorable results from clinical trial patients: how much of the eﬀect is the patient’s inherent response to treatment and how much is attributable to the bioprinted product itself? A valid and comprehensive means of evaluating the eﬀect of bioprinted interventions need to be formally deﬁned prior to commencing any clinical trials of value in this area. Indeed, the only trials of tissue engineered constructs to date have been in patients with terminal disease, where such “last resort” options are often considered “more ethical,” despite the uncertainty of complications. Examples include the use of skeletonized trachea from cadaveric sources, seeded with patient mesenchymal stem cells for use in surgery (29). In these instances, the key to acquiring ethical approval was in the portrayal of the patients’ clinical urgency. Describing the trial of a tissue engineered trachea as the last resort option and as a ﬁnal chance at a lifesaving intervention facilitated the acquisition of ethical approval to implement the treatment in patients (2, 30). This approach was eﬀective in driving an incremental step in translational bioengineering but is a shortcut that presents signiﬁcant limitations. The concept of institutional readiness is a social sciences concept which in essence is a “measure of the capacity and willingness of organizations and inter-organizational structures to adopt, respond to and utilize novel technologies” (10). The signiﬁcance of institutional readiness is that it may impact the technology readiness scale. This may be particularly stark when extrapolated to regenerative medicine interventions: absence of a clear structure within which regenerative medicine interventions can be implemented into health services obstructs the pathway from inception to clinical and commercial success, with the potential to deter investors (10). Institutions who will be responsible for the delivery of regenerative therapies must also display the readiness to cope with the demand for these services. This is likely to include logistical considerations including adequate transport, storage and facilities near to patient for GMP-licensed manufacturing (10). Regulatory Challenges Regulation of these products is another challenge. The high degree of personalisation in construct shape and genetic material renders bioprinted tissue a “custom made device.” The inclusion of biological material complicates the picture, and governing bodies worldwide such as the FDA are failing to keep abreast of the rapidly developing ﬁeld of bioprinting, with currently unclear guidance and regulations for such technology (13). The challenge with regenerative medicine and tissue engineered technologies and their components are their classiﬁcation and ultimate regulation in all facets of design, production, handing. The seemingly endless innovation and advancement of these technologies illustrates the intersection of a number of diﬀerent pathways covering a broad taxonomy of perceived utility, November 2020 \| Volume 7 \| Article 609836 3 3D Bioprinting: Future of Surgery Jovic et al. (31). Indeed the inadequacy of preclinical evidence in this instance, reiterates the importance of a robust foundation of scientiﬁc and clinical validity prior to clinical implementation (3). Other challenges in designing clinical trials include the fact that patients cannot withdraw post-implantation, and consent for trial inclusion is challenging where the extent of complications is uncertain. A perceived beneﬁt of three dimensional structures is that at least a degree of reversibility exists in their ability to be excised if problematic, whereas injectable stem cell and gene based therapies may prove extremely challenging, if not impossible, to reverse (13). and reliable production has posed a number of challenges for regulators and applicants alike. The majority of these innovations are being produced within academic institutions rather than commercial enterprises (26). The challenges therefore must be addressed by clearer communication between regulatory bodies and organizations seeking to produce and market TEPs for clinical use. The EU regulatory committees recognize the challenges posed by the complex nature of these novel technologies and need for development of bespoke guidance as new challenges arise. In the UK, regulators such as the MHRA should be engaged early to facilitate the development of processes and pathways which will ultimately meet standards required to scale tissue engineered constructs for both clinical trials and commercial manufacture. Outside the healthcare setting, incremental advancements in three-dimensional printing technology are yielding more aﬀordable and compact printing systems at an astonishing rate. This rapid evolution indicates three dimensional printers may eventually become household items, much like conventional inkjet printers (27). Technological and Institutional Readiness Should bioprinted technologies surpass the obstacles of clinical proof of concept, widespread uptake of the technology presents a further set of signiﬁcant challenges. The pathway from conception to implementation in patients can be mirrored through the concept of the “Technology Readiness Scale” (32) (Figure 2). In the ﬁeld of bioprinting, much of the current research exists in the TRL1-4 stages; in vitro experimentation with the optimisation of scaﬀold and cell source combinations, bioinks and 3D printing methods and construct analysis (33). There are a handful of groups worldwide who report the use of 3D bioprinted cartilage, bone, skin and vessel constructs in animal models (33) and occasional cases of 3D printing technology being used to make biological constructs in humans (34, 35). In addition to pursuing the appropriate steps of technological readiness as outlined in Figure 2, it is becoming increasingly apparent that “institutional readiness” will be of equal importance in ensuring translation of tissue engineered products into routine clinical practice (10). Regulatory Challenges With these ambitious aspirations however, come fears of the power of such technology being widely accessible. Concerns of unregulated and “DIY” home use may facilitate bioterrorism (28) and unregulated surgical practices, much like the current epidemic of unregulated injectables such as botulinum toxin and ﬁllers. Frontiers in Surgery \| www.frontiersin.org Ethical Challenges Additionally, facilities will be required for the acquisition of donor and autologous tissue for bioengineering and bioprinting which may include integration with the blood and transplant services as an established clinical body for handling tissue and preparing recipients (36). One of the keys to translation will be the engagement and active role of clinicians in ensuring the developments from basic science regenerative medicine and bioprinting research is ethically and clinically viable for widespread use in patients. One of the ways in which this might be expedited is through the use of surgically led, rather than research-led units (30). The caveat to such advancements is the potential to generate uncontrolled and unethical practice. This is particularly starkly demonstrated by the Macchiarini scandal, where the outcomes of the synthetic trachea implantation were falsely augmented November 2020 \| Volume 7 \| Article 609836 Frontiers in Surgery \| www.frontiersin.org 4 Jovic et al. 3D Bioprinting: Future of Surgery FIGURE 3 \| Anticipated trajectory of bioprinting for surgical applications. Major developments in the uses of 3D bioprinted tissue are expected over the next 10–15 years, initially focussing on simple tissue models for drug and cosmetic testing, followed by an increasing number of animal and clinical trials of 3D bioprinted tissue over the next 10 years. Success in these platforms is likely to pave the way for more complex 3D bioprinted constructs such as organs to make an appearance in clinical trials. FIGURE 2 \| Translation of emerging medical technology into clinical use through the Technology Readiness Level model (32). FIGURE 3 \| Anticipated trajectory of bioprinting for surgical applications. Major developments in the uses of 3D bioprinted tissue are expected over the next 10–15 years, initially focussing on simple tissue models for drug and cosmetic testing, followed by an increasing number of animal and clinical trials of 3D bioprinted tissue over the next 10 years. Success in these platforms is likely to pave the way for more complex 3D bioprinted constructs such as organs to make an appearance in clinical trials. FIGURE 2 \| Translation of emerging medical technology into clinical use through the Technology Readiness Level model (32). What Are the Anticipated Developments in 3D Bioprinting in the Next 20 Years? As a biofabrication technology, bioprinting encompasses a combination of software, hardware and wetware processes to enable both high throughput and precise placement of cells, biomolecules and biomaterials in a spatially controlled manner. These properties render bioprinting an ideal technology to replicate native living organoids, tissues and organs, “printing a living environment” for both translational medicine and research purposes. It applies the core doctrines of tissue engineering research in which tissue architecture is emulated through the optimal selection of cell, scaﬀold and growth factor combinations (1), potentiated by the ability to customize, automate and replicate the end tissue engineered product (33). long positioned at the head of the market. Their repertoire rapidly expanded to include printed kidney and liver tissue models for research purposes in addition to 3D skin models for cosmetic testing. However, as with many new technological advancements, the need for signiﬁcant investment front loaded in research and development alongside infrastructure development often creates additional expectation. Whilst overall market investment matched initial hype surrounding the technology, perceived lack of progress resulted in a divestment and directional change for the company in 2019. Twelve years after their 2007 bio- printing debut their CEO Taylor Crouch announced a directional change citing lack of suﬃcient resources to handle the challenges presented by the “variability of biological performance and related duration of potential beneﬁts” of its lead programme. For many this illustrated one of the key issues in the bioprinting market regarding long term investment balanced against market expectations of a return in a timely fashion. Despite taking a hit Stage 2: 3D Bioprinting of Simple Tissues g g It has been predicted that the early stages of using bioprinted products for implantation will occur in the early 2020s (38). It is speculated that the advent of bioprinted implants will spark the emergence of bioprinted tissue for use in regenerative medical and implant-based therapies over the course of the next decade (38). Reconstructive surgery concerns the restoration of form and function to patients aﬀected by congenital abnormalities, trauma, malignancy and burns. In many cases, this population would beneﬁt from relatively small amounts of tissue to restore their form or function. The current treatment options rely heavily on the use of autologous donor tissue to improve the defect. The cost of such a treatment is the creation of a defect or scar elsewhere on the body. Bioprinting oﬀers the potential to evade donor sites and the associated complications of their use with the potential to be life changing. Connective tissues are an achievable medium-term goal for bioprinting. Structures such as cartilage are avascular, aneural and devoid of the extensive cell-cell connections that underpin solid viscera (39). As such, they have been a subject of increasing interest from scientists, clinicians, industry and investors alike (40). Despite global eﬀorts to advance connective tissue bioengineering, there remains a lack of successful translation. There remains a degree of dispute regarding optimal cell sources and scaﬀolds (41), means of ensuring adequate vascularization (42), characterization and proof of safety prior to implantation (43), and durability in animal models (41). Current high-proﬁle failures in tissue engineering cartilage (in part due to the clinical models in which they were used) highlight the current shortcomings. 3D Printed Implants and Prosthetics Firstly, with open source 3D printing ﬁles compatible with most printer-based technology available on the internet, there is an increased availability of “blueprints” from which clinicians can choose and initiate printing of a 3D product within minutes (49). By decentralizing the manufacturing process and circumventing transportation and logistical barriers that delay treatment clinicians would have greater access to a range of print ﬁles for their patients. For example, upon diagnosis of severe aortic stenosis, the blueprint for a new aortic valve could be downloaded and printed within minutes. Bioprinting also means that the valve could be printed with bioinks such as collagen, that more closely emulate native tissue valves than plastics (50), or even with the patient’s native valvular cells to truly personalize the product (51). To a degree, similar processes already exist for dental ﬁllings and neurosurgical cranial plugs (52). This potential to rapidly download and produce 3D products based on tissue blueprints could even be extrapolated to simple, generic prosthetics. However, the availability of medical images such as CT scans and increasingly seamless integration into 3D printing technologies means true customization and anatomical matching is an achievable reality. In maxillofacial surgery, a combination of contour models, guides, splints and implants have been extensively generated through 3D printing with an average production time of under 24 h (53). This technology could be extrapolated to joint replacements, pacemakers, cochlear implants and other implantable medical devices. Furthermore, three dimensional printing enables the speciﬁc seeding of pharmaceutical agents such as antimicrobial, immunomodulatory or analgesic agents during the printing process with the potential to generate a new class of bioactive medical implants (54). Stage 1: 3D Bioprinting for Drug and Cosmetic Testing Organovo were the ﬁrst company to enter the 3D bioprinting sphere in 2007, oﬀering functional bioprinted blood vessels. As early adopters of the technology, the San Diego based biotech paved the way for creation of 3D bioprinted organoids and were November 2020 \| Volume 7 \| Article 609836 Frontiers in Surgery \| www.frontiersin.org 5 3D Bioprinting: Future of Surgery Jovic et al. neovascularisation to enable the survival of the tissue construct (46). Mastery of composite tissue engineering is likely to precede the ability to produce functional organs, which in addition to surmounting the challenges of multiple cell types and vascularisation will require organization of tissue into an organ-speciﬁc topography and mimicry of the complex endocrine and physiological roles served by solid organs (47). Furthermore, detailed assessments of the safety, longevity and biocompatibility of smaller bioprinted constructs will need to ﬁrst be veriﬁed and optimized prior to the irreversible action of transplanting a large construct with physiological roles such as a solid organ. in share price, a recent merger with Tarveda Theraputics has seen the company change direction with a focus on precision oncology medicines. In spite of this strategic change, the value of 3D bioprinted models for drug testing and basic science research has signiﬁcant value and remains a key area of research investment. Currently used cell lines and animal models often fail to emulate the behavior of human tissues and underpin many of the failures of translation to human clinical drug trials. Currently, several drug companies are printing tissue for use in drug testing: Aspect Biosystems have been developing bioprinted lung tissue for this purpose since 2015 (37). Is There a Role for 3D Bioprinting in Personalized Medicine? 3D bioprinting oﬀers the opportunity to manage disease through personalized treatments and to produce therapeutics on an industrial scale (48). Bioprinting is likely to augment personalized healthcare through eﬃcient coupling of diagnosis to intervention, translating patient speciﬁc images into tailored implants and prosthetics, advancing cell and gene-based therapies and regenerative medicine. Frontiers in Surgery \| www.frontiersin.org Biologically Active 3D Printed Implants Biologically Active 3D Printed Implants that work in synergy to maintain the structural and functional components of the organ. The complex interplay between bile ducts, hepatocytes, vasculature and connective tissue act synergistically to serve digestive, endocrine and hematological roles. Although tissue engineering and 3D bioprinting may ultimately be able to emulate the complex topography and function of solid viscera, replication of this interplay in vitro will be fraught with technical and biological diﬃculty (61). The ability to incorporate cells into the 3D constructs would additionally transform the ability to personalize pharmaceutical and disease management. Currently, several drug companies are printing tissue for use in drug testing: Aspect Biosystems have been developing bioprinted lung tissue for this purpose since 2015 (37). 3D bioprinting research has also been expanded to address the management of diseases such as type 1 diabetes, through the 3D printing of human beta-like cells capable of glucose mediated insulin secretion (55, 56). The high resolution of bioprinting conveys the advantage of enabling the deposition in nano to microscale array to mirror histological and macroscopic morphology of diﬀerent tissues (62). In the post-processing phase, bioreactors oﬀer a dynamic environment for tissue maturation to occur, though precautions must be implemented to minimize the risk of tissue damage during the maturation process (63). Bioprinting is currently the best suited biofabrication method to achieve the required porosity, geometry and interconnectivity of complex structures such as solid viscera and is likely to underpin major advancements in the ﬁeld over the next 20 years. To date, there have been a number of ﬁrst in human implantations of tissue- engineered constructs, not all of which have been successful, highlighting the need for robust preclinical evidence and high- quality clinical trials in this ﬁeld to ensure patient safety (Table 1). The next step in revolutionizing personalized prosthetics is a like-for-like replacement of the defective tissue with tissue engineered constructs. 3D bioprinting oﬀers the ability to print constructs such as ears in the exact shape of the patients missing auricle using a bioink conducive of de novo cartilage formation (44). As such, when laced with the patient’s own cartilage cells, an exact cartilaginous match of the contralateral ear could be generated. The mastery of 3D bioprinting would merge the accuracy of printed medical prosthetics with the beneﬁts of autologous reconstruction to yield a replacement that would be unparalleled in its resemblance to native tissue (33). Is 3D Bioprinting the Answer to Organ and Tissue Transplantation? p g Market Overview: Base Technology gy The exponential development of the 3D bioprinting market in both the academic and commercial settings is largely due to the successful development and adaptation of the base technologies ﬁrst pioneered in the 1980s. The 3D printing and additive manufacturing (AM) industry surpassed forecasted growth expectations and in the “2018 Wohlers Report” they cited a 21% growth of AM products and services world- wide, currently valuing the market at $7.336 Billion, $1 Billion ahead of initial projections. Key investments in Research and Development and the signiﬁcant development of entry level platform technologies saw an estimated 520,000 desktop 3D printers under $5000 sold in just 2 years, with Ultimaker, MakerBot and Aleph Objects contributing to an estimated $500 million made from just this technology alone (68). Regardless of the product oﬀering companies continue to use and reﬁne one of the four main print-head technologies; extrusion, laser-induced, inkjet and microvalve. Extrusion (syringe based) technology continues to command the greatest market share due to its aﬀordability, ease of adaptation and broad applications. p g There is an increasing clinical need for organ and tissue replacement therapy (57). In 2016–2017, 6389 patients were awaiting an organ transplant in the UK, yet only 3712 donor transplants were available during this period (58). These statistics underpin the sobering paradigm of modern healthcare: the availability of resources is an inadequate solution to the scale of the clinical problem. In addition to the stark shortage of suitable organ donors, receipt of an organ transplant requires a lifetime of immunosuppressive medication whilst still retaining a lifetime risk of rejection and immune-mediated diseases. Ultimately, these patients may ﬁnd themselves in need of an additional organ transplant or even facing death. The promise of bioprinting is to truly personalize tissue engineering: using a patient’s own cells and genetic material to generate a replacement viscera in a shape and structure that matches their own anatomy. Biologically, this obviates the need for HLA matching, the risk of acute rejection and facilitates long-term integration of the organ into the recipient. Structurally, it enables the combination of multiple tissue types arranged precisely in the tissue’s native microarchitecture and microenvironment to provide an organ that is truly the patient’s own. Biologically Active 3D Printed Implants As previously discussed, the ultimate potential of 3D bioprinting is the production of patient speciﬁc body parts such as organs and limbs, with the capability of revolutionizing personalized medicine and surgery. Bioprinting Market There are numerous market forces which have been instrumental in driving up the compound annual growth rate (CAGR) with global market projections of between 26 and 36% in the next 5–10 years (69). Advancements in production technology and biomaterials innovation alongside increasing cost and regulatory complexity in the manufacture and testing of pharmaceuticals and cosmetics saw the ﬁrst wave of companies enter the market in 2014 oﬀering novel printers and tissue speciﬁc assays. Stage 3: 3D Bioprinting of Complex Tissues and Organs g The natural progression thereafter would be to engineer composite tissues. Clinically, defects in need of surgical reconstruction often consist of multiple cell types, for example cartilage perichondrium and skin in ear reconstruction (44), or bone, periosteum and mucous membranes in cleft palate repair. Although signiﬁcant advancements are being made in the engineering of single tissue types, composite tissue engineering adds an additional layer of complexity (45). Production of multilaminar constructs requires a combining of scaﬀolds, cell source and environments that accommodate each of the intended tissue types. Furthermore, the maintenance of an appropriate interface between the tissue layers, such as in skin, presents a new obstacle, compounded by the need for November 2020 \| Volume 7 \| Article 609836 Frontiers in Surgery \| www.frontiersin.org 6 3D Bioprinting: Future of Surgery Jovic et al. Frontiers in Surgery \| www.frontiersin.org Future Developments The diverse applications of bioprinting technology have already been demonstrated on a global scale, leading to the production of novel constructs from vessels and composite tissue, to organoids and complex cellular and tissue models for drug, cosmetic and experimental testing. The 3D bioprinting market has seen oﬀshoot companies set up to corner a speciﬁc sub-set of the production and manufacturing of complex 3D printed tissues; from desktop 3D bioprinters and bioinks, to scaﬀolds pre-loaded with and without growth factors generating a market value in the $US billions. The diversiﬁcation of this technology and its associated components demonstrate the key issue with this extraordinary technology and potential diﬃculty in harnessing its true potential; the lack of “end to end” visibility by any one agency. The creation of complex human tissue arrays and organoids has not gone unnoticed by researchers around the world looking to better create 3D models of complex diseases such as cancer. The production of 3D vascularised tumor models “organ on a chip” has been created to better understand the complex interplay between cancer and multi-organ metastasis and paracrine signaling mechanisms in the regulation of breast cancer metastasis (70). This novel utilization of 3D printing has the potential to advance our understanding of complex disease and develop novel personalized treatments for diseases such as caner which currently account for one in seven deaths worldwide (71). The continued development and application of this base technology promises to 1 day make the creation of bespoke tissue engineered constructs and “made to order” solid complex organs a reality. The technological revolution in the last two decades has seen the development of intelligent bio-inks, reﬁnement of printing techniques and production of novel biomaterials to facilitate the creation of custom scaﬀolds to support cellular growth (72). Since 2014 a number of 3D bioprinting companies, start-ups and R&D spinouts have entered the market contributing to the commercial development of this novel technology and creating a projected market value based on the early success and novel application of 3D bioprinted products (38). With a market value estimated at around $680 Million in The translation of 3D printed constructs into clinical practice is challenging. The optimisation of the translational pathway demands concerted eﬀorts from scientists, engineers and clinicians, contextualized within an infrastructure in which an eﬀective supply chain exists. The Role of Organ Biofabrication As the market develops there appears to be a divergence with companies such as EnvisionTec, GeSIM, Cellink, and BioBots choosing to focus on printer technology and associated consumables, whereas Organovo focus on the production of cellular products and tissue assays. Maintaining its position as the market leader and ﬁrst ever publicly traded bioprinting company; Organovo initially oﬀered a small number of human tissue assays including liver and kidney for drug testing and pre-clinical research and has since developed a custom tissue partnership oﬀering the opportunity to develop unique tissue models and assays speciﬁc to an individual company or researchers needs. In 2014 Organovo in partnership with L’Oreal, developed 3D bioprinted skin models to get ahead of the EU directives banning the testing of cosmetics on animals (13). CONCLUSIONS 3D printing and bioprinting has the potential to be the single biggest technological disruptor to the current model for design and delivery of healthcare and research in this century. The incorporation of human cells and biocompatible materials into 3D printing practice is set to deliver a paradigm shift in the application of 3D printing for surgery, oﬀering the potential to 3D print living tissue and organs. The promise to 3D print de novo body parts, obviate the need for organ transplantation and to replace the role of animals in the development and testing of novel drugs, means patients could potentially have access to a bespoke treatments at every point in their healthcare journey. The Role of Organ Biofabrication g Despite in vitro and in vivo studies supporting the feasibility of tissue engineering for use in a multitude of clinical scenarios (59, 60) obstacles remain that are hindering clinical translation. At the top end of the spectrum, solid organs such as the liver display a complex three-dimensional array of diﬀerent tissue types November 2020 \| Volume 7 \| Article 609836 Frontiers in Surgery \| www.frontiersin.org 7 3D Bioprinting: Future of Surgery Jovic et al. TABLE 1 \| Applications of tissue-engineered constructs in humans [adapted from Al-Himdani et al. (44)]. Organ/tissue No. of patients Cell source Outcomes References Bladder 7 Bladder urothelial and muscle cells Improved volume and compliance with no metabolic consequences at mean 46 months follow-up (64) Trachea 1 Recipient MSCs Functional airway with a normal appearance and mechanical properties at 4 months, recent controversy (29) Urethra 5 Muscle and epithelial cells Maintenance of wide urethral calibers without strictures, normal architecture on biopsy at 3 months following implantation (65) Nasal cartilage 5 Autologous nasal chondrocytes Good structural stability and respiratory function after 1 year (66) Vaginal organs 4 Vulval biopsy—epithelial and muscle cells Tri-layered structure on biopsy with phenotypically normal smooth muscle and epithelia with follow-up up to 8 years (67) Auricular cartilage 5 Autologous auricular chondrocytes Evidence of cartilage formation at 6 months in 80% of patients, structural deformation noted in most cases (35) 2016, industry reports project growth to reach $1.9 Billion by 2027 (73). As the market develops there appears to be a divergence with companies such as EnvisionTec, GeSIM, Cellink, and BioBots choosing to focus on printer technology and associated consumables, whereas Organovo focus on the production of cellular products and tissue assays. Maintaining its position as the market leader and ﬁrst ever publicly traded bioprinting company; Organovo initially oﬀered a small number of human tissue assays including liver and kidney for drug testing and pre-clinical research and has since developed a custom tissue partnership oﬀering the opportunity to develop unique tissue models and assays speciﬁc to an individual company or researchers needs. In 2014 Organovo in partnership with L’Oreal, developed 3D bioprinted skin models to get ahead of the EU directives banning the testing of cosmetics on animals (13). Frontiers in Surgery \| www.frontiersin.org November 2020 \| Volume 7 \| Article 609836 REFERENCES Cold Spring Harb Perspect Med. 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Angiomyeloproliferative lesions following autologous stem cell therapy. J Am Soc Nephrol. FUNDING encourage its integration into mainstream surgical practice. With incremental advances in 3D printing and bioprinting expected over the next century, the impact on the future of surgery could be transformational. The Reconstructive Surgery & Regenerative Medicine Research Group was supported by the Medical Research Council (Grant Number: MR/N002431/1) (ZJ), Microtia UK (TJ and IW), Action Medical Research (Grant Number: GN2782) (TJ), the Vocational Training Charitable Trust Foundation (TJ), the Welsh Clinical Academic Training Programme (TJ, ZJ, and EC), the Royal College of Surgeons (TJ, EC, ZJ, and IW), and the American Association of Plastic Surgeons/European Association of Plastic Surgeons Academic Scholarship (IW). AUTHOR CONTRIBUTIONS IW, ZJ, and TJ conceived the idea for the manuscript. TJ and EC drafted the manuscript with signiﬁcant contribution from ZJ and IW. 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https://openalex.org/W381409476	https://isprs-archives.copernicus.org/articles/XL-5-W6/51/2015/isprsarchives-XL-5-W6-51-2015.pdf	English	null	HUMAN ACTION POSELETS ESTIMATION VIA COLOR G-SURF IN STILL IMAGES	The international archives of the photogrammetry, remote sensing and spatial information sciences/International archives of the photogrammetry, remote sensing and spatial information sciences	2,015	cc-by	7,492	1. INTRODUCTION noise. Shakhnarovich et al (Shakhnarovich et al., 2003) proposed interesting hashing-based search technique for pose estimation relevant to pose examples in a large database. A fast pruning method based on shape contexts in order to speed up the search for similar body poses was presented by Wang et al. (Wang et al., 2006). High computational cost of searches in the high-dimensional spaces inside large datasets is the major drawback of example-based methods. Recognition of human active actions is employed in many tasks of computer vision, e.g., video surveillance, human detection, activity analysis, scene analysis, image annotation, image and video retrieval, augmented reality, human-computer interaction, among others. Conventional methods of human action recognition use motion information in videos (Laptev and Lindeberg T, 2003, Zhen et al., 2013), which can be applied for object capturing and then for trajectory motion analysis as the temporal component in human action classification. During action classification, the spatial component plays a significant role that makes reasonable to develop methods for human action recognition in still images. The pictorial structure-based methods represent poses as the cues using prior information of a human body structure (Felzenszwalb and Huttenlocher, 2005). In this approach, the histogram-based methods prevail. It may be circular histograms of spatial and orientation binning (Ikizler et al., 2008) or the most popular Histogram of Oriented Gradients (HOG) (Dalal, and Triggs, 2005) with multiple modifications. The last research was the pioneer investigation in pose descriptor construction based on non-negative matrix factorization. The action classes were represented by the HOGs of pose primitives with following simple histogram comparison for action recognition. This technique works well in typical cases but it fails in occlusions or significant changes of camera viewpoints. To overcome these problems, Delaitre et al. (Delaitre et al., 2010) proposed a Bag-of-Features (BoF) approach for human action recognition in still images in combination with Support Vector Machine (SVM) classification. They combined the statistical and part-based representations integrating a person-centric description in cluttered background. Methods used to estimate the human poses can be classified in four categories: model-based, example-based, pictorial structure-based, and poselet-based approaches. The model- based methods predefine a parametric body model and find the pose matching based on labelled extracted features. A graphical model of human-object interactions was developed by Gupta et al. (Gupta et al., 2009) including reach motions, manipulation motions, and object reactions. HUMAN ACTION POSELETS ESTIMATION VIA COLOR G-SURF IN STILL IMAGES M. Favorskaya , D. Novikov, Y. Savitskaya matics and Telecommunications, Siberian State Aerospace University, 31 Krasnoyarsky Rabochy av., Krasnoyarsk, 660014 Russian Federation - (favorskaya, novikov_dms)@sibsau.ru, juliklapa@yandex.ru Institute of Informatics and Telecommunications, Siberian State Aerospace University, 31 Krasnoyarsky Rabochy a 660014 Russian Federation - (favorskaya, novikov_dms)@sibsau.ru, juliklapa@yandex.ru Commission WG V/5, WG III/3 KEY WORDS: Human Action, Poselets, Gauge-SURF, Random Forest, Still Image ABSTRACT: Human activity is a persistent subject of interest in the last decade. On the one hand, video sequences provide a huge volume of motion information in order to recognize the human active actions. On the other hand, the spatial information about static human poses is valuable for human action recognition. Poselets were introduced as latent variables representing a configuration for mutual locations of body parts and allowing different views of description. In current research, some modifications of Speeded-Up Robust Features (SURF) invariant to affine geometrical transforms and illumination changes were tested. First, a grid of rectangles is imposed on object of interest in a still image. Second, sparse descriptor based on Gauge-SURF (G-SURF) invariant to color/lighting changes is constructed for each rectangle separately. A common Spatial POselet Descriptor (SPOD) aggregates the SPODs of rectangles with following random forest classification in order to receive fast classification results. The proposed approach was tested on samples from PASCAL Visual Object Classes (VOC) Dataset and Challenge 2010 providing accuracy 61-68% for all possible 3D poses locations and 82-86% for front poses locations regarding to nine action categories. Corresponding author The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XL-5/W6, 2015 Photogrammetric techniques for video surveillance, biometrics and biomedicine, 25–27 May 2015, Moscow, Russia The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XL-5/W6, 2015 Photogrammetric techniques for video surveillance, biometrics and biomedicine, 25–27 May 2015, Moscow, Russia The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XL-5/W6, 2015 Photogrammetric techniques for video surveillance, biometrics and biomedicine, 25–27 May 2015, Moscow, Russia This contribution has been peer-reviewed. doi:10.5194/isprsarchives-XL-5-W6-51-2015 1. INTRODUCTION Such models are often built on silhouette-based representation of body parts or edge information. The main disadvantage deals with difficulties in design of parametric body model and pose sub-models. The example-based methods do not use a global modelling structure and store a set of images with corresponding pose descriptions. In this framework, two problems appear as relevant descriptors and fast search. Three shape descriptors – Fourier descriptors, shape contexts, and Hu moments were compared by Poppe and Poel (Poppe and Poel, 2006) for representation of human silhouettes. They experimented with deformed silhouettes robustness to body sizes, viewpoint, and The poselet-based methods provide rich information about locations of body parts. They are built on 3D pose images. The poselets were introduced by Bourdev and Malik (Bourdev and Malik, 2009) for person detection in natural framework. The detailed literature review of methods from the fourth category is This contribution has been peer-reviewed. doi:10.5194/isprsarchives-XL-5-W6-51-2015 This contribution has been peer-reviewed. doi:10.5194/isprsarchives-XL-5-W6-51-2015 This contribution has been peer-reviewed. doi:10.5194/isprsarchives-XL-5-W6-51-2015 51 The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XL-5/W6, 2015 Photogrammetric techniques for video surveillance, biometrics and biomedicine, 25–27 May 2015, Moscow, Russia The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XL-5/W6, 2015 Photogrammetric techniques for video surveillance, biometrics and biomedicine, 25–27 May 2015, Moscow, Russia The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XL-5/W6, 2015 Photogrammetric techniques for video surveillance, biometrics and biomedicine, 25–27 May 2015, Moscow, Russia presented in following section. Better pose descriptor means a wide invariance to various geometric transforms, lighting, viewpoints, and image warping in general case. This difficult task is in the field of current and following investigations. Our contribution deals with the proposed color Gauge-SURF with selection of special imposed grids in human body image for human action recognition. The original Gauge-SURF was extended by invariance to color and lighting changes. of a person. Some approaches extract features in areas within or surrounding the human bounding boxes. Delaitre et al. (Delaitre et al., 2010) defined a person setting in each image in one and a half time more that the sizes of human bounding box. Then these regions are resized up to 300 pixels (in larger size) and analyzed using low-level features. Some methods extract contour information of human body and body parts from still images. Wang et al. 2. RELATED WORK Yang et al. (Yang et al., 2010) developed a coarse example- based poselets representation, when each body part may have more than 20 poselets concerning to different body poses. They constructed a set of four corresponding body parts L = {l0, l1, ..., lk–1} denoting the upper-body, legs, left-arm, and right-arm. Often a graph is a good model to represent the relations between different body parts. Raja et al. (Raja et al., 2011) constructed a graphical model containing six nodes: the action label and five body parts correspond to head H, right-hand RH, left-hand LH, right-foot RF, and left-foot LF. The links between nodes encode action-dependent constraints on the relative positions of body parts. The poselet as a subject of interest is 2D still non-segmented image of configured body parts in 3D space (head, shoulders, arms, torso, legs) capturing a part of neighbouring background. Thus, the poselet of single pose is a collection of 2D still images received from various shooting viewpoints, in different scales and lighting conditions. Sometimes, the presence of articulation makes the pose estimation harder. Although the pose estimation is used for human action recognition, one may consider the pose estimation as a separate task of computer vision. Figure 1 depicts some examples of human actions. Figure 1. Human pose examples In many scenarios, a person relates with other objects, e.g. phone, ball, animal, etc. These objects serve as a source of reliable information about category of human action. Some approaches analyse individual objects separately while other methods consider them as a scene context. As a result, some methods were developed as a Human Object Interaction (HOI), for example, weakly supervised method proposed by Prest et al. (Prest et al., 2012). 1. INTRODUCTION (Wang et al., 2006) exploited the overall coarse shape of human body as a collection of edge points obtained via Canny edge detector. Then the received features were classified and categorized into different actions. Also semantic features can be used to describe the actions in images with the human body (Yao et al., 2011). The attributes were related to verbs in a human language and remained visual words from annotation system. In the following, Section 2 gives a brief review of high-level and low-level evaluations of poselets. The G-SURF background is presented in Section 3. The proposed methodology of poselets estimation is explained in Section 4. The experimental results are presented and analyzed in Section 5. The paper is concluded in Section 6. 2.2 Low-level Evaluation The high-level evaluation is usually based on various low-level scores. The DSIFT features are extracted from many image patches with following clustering to obtain a limited number of “keywords”, which are grouped in a codebook. Many methods use the DSIFT features due to their possibility for direct classification of human actions. One can mention the researches of Delaitre et al. (Delaitre et al., 2010), Yao et al. (Yao et al., 2011), etc. Figure 1. Human pose examples Since only the spatial information is available in still images, one can represent information as high-level cues and low-level cues/features. Description of human body, body parts, action- related objects, human object interaction, and scene context are included in high-level cues. Typical low-level features are a Dense sampling of Scale Invariant Feature Transform (DSIFT), HOG, Shape Context (SC), GIST, and some other features. Short surveys of high-level and low-level evaluation methods are situated in Sections 2.1 and 2.2, respectively, while existing 3D-based poselet methods are discussed in Section 2.3. The HOG descriptor is very popular for pedestrian detection (Dalal and Triggs, 2005). The HOG descriptor counts the occurrences of discrete gradient orientations within a local image patch similar to the edge orientation histogram, the SIFT, and the SC. The SC is useful to detect and segment the human contour; however, this technique is crucial for high-level cue representation of human body silhouettes. The spatial envelop or GIST was proposed by Oliva and Torralba (Oliva and Torralba, 2001). A set of spatial properties in a scene can be computed by the GIST method, which provides the abstract category representations of a scene based on integrated background information. This approach has been used by Gupta et al. (Gupta et al., 2009), Prest et al. (Prest et al., 2012), among others. One can mention the development of other approaches based on SURF (Bay et al., 2008), Circular 2.3 Towards to 3D Representation                             σ , , σ , σ , σ , 1 σ , , σ , ; σ , , σ , σ , σ , 1 σ , , σ , 2 2 2 2 p p p p p p p p p p p p x y y x y x y x L L L L x L y L v L L L L y L x L w                                 (3) Some methods deal with detection of view-independent objects using 3D object models. Glasner et al. (Glasner et al., 2011) used a viewpoint estimation method for rigid 3D objects from 2D images by voting method for efficient accumulation of evidence. This method was tested on rigid car data. Fidler et al. (Fidler et al., 2012) developed a method for localizing objects in 3D space by enclosing them within tightly oriented 3D bounding boxes. This model represents an object class as a deformable 3D cuboid by anchors of body parts in 3D box. Hejrati and Ramanan (Hejrati and Ramanan, 2012) developed a two-stage model, when, first, a large number of effective views and shapes are modelled using a small number of local view- based templates and, second, these estimates are refined by an explicit 3D model of the shape and viewpoint. (3) where w = a gradient vector where w = a gradient vector where where v = a perpendicular direction vector  = a kernel's standard deviation or scale parameter L(p, ) = a convolution of image I(p) with 2D Gaussian kernel g(p, σ) Another issue is a computational speed required for view- independent object detection because the most methods generate the classifiers at multiple locations and scales. Sometimes, the shared features are extracted to reduce a number of classifiers and the runtime complexity correspondingly. Razavi et al. (Razavi et al., 2010) used an extension of Hough- based object detection and built a shared codebook by jointly considering several viewpoints. Tosato et al. 2.3 Towards to 3D Representation All variety of poselet-based methods may be classified according to various criteria. A viewpoint dependence/ independence in 2D/2D spaces, respectively, can be considered the main criterion. Bourdev and Malik (Bourdev and Malik, 2009) were the first, who formulated the task of human pose estimation and recognition as 3D object representation. They constructed the body part detectors trained from annotated data of joint locations of people and based on patches similarities. As a result, the poselet activation vector consisting of poselets inside the bounding box was introduced. The SVM classifier was used to recognize these patches. The distribution of these joints and personal bounding boxes can be obtained to each poselet. A convolution Lxx(p, ) is determined by Equation 2:     σ * σ , 2 2 g x I Lxx    p p (2) (2) (2) and similarly for Lxy(p, ) and Lyy(p, ) along diagonal and OY directions, respectively. Alcantarilla et al. (Alcantarilla et al., 2013) developed a novel family of multi-scale local feature descriptors called as Gauge-SURF (G-SURF). In this case, every pixel in the image is described by 2D local structure. The multi-scale gauge derivatives are invariant to rotations and shifts. Additionally, they describe the non-linear diffusion processes. The use of G-SURF makes blurring locally adaptive to the region so that noise becomes blurred, whereas details or edges remain unaffected. Such local structures are described by Equation 3: Russakovsky et al. (Russakovsky et al., 2012) developed an Object-Centric spatial Pooling (OCP) approach for detection an object of interest. The local OCP information is used to pool the foreground and background features. Khan et al. (Khan et al., 2013) applied a comprehensive evaluation of color descriptors with combination of shape features. where p = (x, y)T = a point in an image I  = a scale factor where p = (x, y)T = a point in an image I  = a scale factor Histogram of Oriented Rectangles (CHORs) (Ikizler et al., 2008), Adaboost classifiers (Gupta et al., 2009). Lxx(p, ) = a convolution of an image I(p) in a point p with a Gaussian second order derivative along direction OX 2.3 Towards to 3D Representation (Tosato et al., 2010) modelled a human image as a hierarchy of fixed overlapping parts. Each part was trained using a boosted classifier learned using Logicboost algorithm. Velaldi et al. (Velaldi et al., 2009) introduced a three-stage SVM classifier combining linear, quasi-linear, and non-linear kernels for object detection. Using gauge coordinates, one can obtain a set of derivatives invariant to any order and scale. The second-order gauge derivatives Lvv(p, ) and Lww(p, ) are in special interest. They can be obtained as a product of gradients in w and v directions and the 2  2 second order derivatives or Hessian matrix provided by Equation 4:                                                                              σ , σ , σ , σ , σ , σ , σ , σ , σ , σ , 1 σ , ; σ , σ , σ , σ , σ , σ , σ , σ , σ , σ , 1 σ , 2 2 2 2 p p p p p p p p p p p p p p p p p p p p p p x y yy yx xy xx x y y x vv y x yy yx xy xx y x y x ww L L L L L L L L L L L L L L L L L L L L L L (4) (4) 2.1 High-level Evaluation A human body image is an important cue in human action recognition, which can be detected automatically or manually labelled. Usually a bounding box is used to indicate a location This contribution has been peer-reviewed. doi:10.5194/isprsarchives-XL-5-W6-51-2015 52 The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XL-5/W6, 2015 Photogrammetric techniques for video surveillance, biometrics and biomedicine, 25–27 May 2015, Moscow, Russia The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XL-5/W6, 2015 Photogrammetric techniques for video surveillance, biometrics and biomedicine, 25–27 May 2015, Moscow, Russia 3. G-SURF BACKGROUND The original SURF defines the determinant of the approximate Hessian matrix in the points, where the determinant has maximum values (Bay et al., 2008). The Hessian matrix H(p; ) is defined by Equation 1:                   σ , σ , σ , σ , σ ; p p p p p H yy xy xy xx L L L L (1)                   σ , σ , σ , σ , σ ; p p p p p H yy xy xy xx L L L L (1) The G-SURF descriptor is based on the original SURF descriptor. Mention some modifications such as Modified Up- right SURF (MU-SURF) descriptor based on Haar wavelets The G-SURF descriptor is based on the original SURF descriptor. Mention some modifications such as Modified Up- right SURF (MU-SURF) descriptor based on Haar wavelets                   σ , σ , σ , σ , σ ; p p p p p H yy xy xy xx L L L L (1) This contribution has been peer-reviewed. doi:10.5194/isprsarchives-XL-5-W6-51-2015 53 This contribution has been peer-reviewed. doi:10.5194/isprsarchives-XL-5-W6-51-2015 53 53 The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XL-5/W6, 2015 Photogrammetric techniques for video surveillance, biometrics and biomedicine, 25–27 May 2015, Moscow, Russia The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XL-5/W6, 2015 Photogrammetric techniques for video surveillance, biometrics and biomedicine, 25–27 May 2015, Moscow, Russia The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XL-5/W6, 2015 Photogrammetric techniques for video surveillance, biometrics and biomedicine, 25–27 May 2015, Moscow, Russia responses and two Gaussian weighting steps, the Center Surround Extremas (CenSurE) for approximation the bi-level Laplacian of Gaussian using boxes and octagons, the Speeded Up Surround Extrema (SUSurE), which is a fast modification of the MU-SURF, and the CenSurE descriptors for mobile devices. These modifications well describe edges, angles, boundaries of unknown objects in an image. However, they poorly concern color information, which is useful in analysis of still images. texture under assumption that such large areas are the body parts. 4. ESTIMATION OF POSELETS The poselets estimation is concerned to identification task, when a number of classes is restricted by a finite set of human poses describing in feature space by some descriptors. The complexity deals with another issue – the great variants of 3D images of a single pose mapping in 2D still images. Nowadays, this problem has not been solved, and many authors develop heuristic algorithms, more or less successful. The structure of bounding boxes, in common case, grid, for poselet detection is discussed in Section 4.1. The proposed color G-SURF family is represented in Section 4.2. Section 4.3 provides a classification procedure based on random forest in order to identify a testing poselet. 3. G-SURF BACKGROUND These areas representing the bounding boxes are the basis of the SPOD constructions, which later are aggregated in a common SPOD for classification. This task is similar to automatic image annotation. Our recommendations deal with the use of J-SEG algorithm or similar ones. If this assumption is not fulfilled, then additional human segmentation methods are required in order to receive the cropped image of human body. Let us suppose that cropped still image of human body is obtained, and six aspect ratios are used including head, torso, left arm, right arm, left leg, and right leg. Each of the arm and leg boxes is composed from two sub-boxes including hands and feet. A number of imposed aspect ratios can be reduced due to human action and human position. Moreover, a number of imposed aspect ratios serve as a weak classifier for human action categorization. For example, the active action “phoning” may include an image of head and any arm while the active action “running” may involve images of torso and both legs. 4.2 The Proposed Color G-SURF Family Under various conditions of color/lighting changes in human pose images, it is important to develop a descriptor invariant to these changes. Our contribution deals with the development of original G-SURF, which is invariant to geometrical (affine) distortions. Family of the proposed color G-SURF descriptors includes the following components. 4.1 Imposed Grid for Poselet Detection For poselet recognition, it is required to capture a salient part of one’s pose from a given viewpoint and impose a set of corresponding rectangle boxes at given orientation, position, and scale. Many authors use the predetermined aspect ratios sometimes with normalization of distance between hips and shoulders. Bourdev and Malik (Bourdev and Malik, 2009) used the poselets with following of aspect ratios 96  64, 64  64, 64  96, and 128  64 pixels. Their algorithm was trained using 300 poselets of each type of pose. Then a model predicting a bounding box for each poselet was fitted. Additionally, the overlapping bounding boxes were considered with overlapping area more 20%. These scores were added to the basic set. The 1200 dimensional vector to estimate the human pose was constructed, besides the poses of head and torso were considered separately. This approach was developed by Ko et al. (Ko et al., 2015), when five aspect ratios such as 96  64, 64  64, 64  96, 64  128, and 128  64 pixels were used in order to consider variations of human poses. These authors modified the selecting algorithm of the action poselets using the modified Hausdorff distance with Epanechnikiv kernel. The descriptor is based on Oriented Center-Symmetric Local Binary Patterns (OCS-LBPs) due to their low computational complexity. Such approach proposes the overlapping of aspect ratios with preliminary rough body parts segmentation. Background is involved in this grid. Often segmentation is implemented manually. The rg G-SURF (rgG-SURF) includes the chromaticity components r and g invariant to scale and light changes. 4.1 Imposed Grid for Poselet Detection The transformed RGB color G-SURF (RGBG-SURF) descriptor is computed for each normalized RGB channel by Equation 7:                                    B B G G R R B G R B G R σ μ σ μ σ μ (7) (7) In this study, the common SPOD in a view of the concatenated histograms (OX and OY) of color G-SURF descriptors in each bounding box is used as a feature for random forest classification. In training stage, each tree T is constructed based on a set of examples Exi of a poselet Exi = (ei, ci), where ei is a poselet example and ci is a class label of a poselet example. The positive poselet examples are marked by a class label ci = 1, and the negative poselet examples gain a class label ci = 0. Samples of the other body parts including a background are concerned to the negative poselet examples. During tree construction, each leaf node L stores the class information CL of the examples. When only positive poselet examples are reached at node N, then CL = 1. Value of CL is proportional to a number of positive poselet examples. A split function is assigned to each non-leaf node in such manner that the uncertainties in class labels ought to be reduced towards leaves. In testing stage, the current element descriptor of a poselet is checked using the created binary trees by sequential comparison with value of split function in each node. where R, G, B = means of the distribution in RGB channels computed in a chosen region of image, respectively R, G, B = standard deviations of the distribution in RGB channels computed in a chosen region of image, respectively The RGBG-SURF is invariant to scale and shift with respect to light intensity (Van de Sande et al., 2009), while a classic RGB histogram is not invariant to changes in lighting conditions The histogram has 45 dimensions. Also one can mention additional color descriptors with some invariance to color/lighting conditions such as the color moment histogram, the hue-saturation descriptor, the color names, the discriminative color descriptor, among others. 4.1 Imposed Grid for Poselet Detection doi:10.5194/isprsarchives-XL-5-W6-51-2015 54 The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XL-5/W6, 2015 Photogrammetric techniques for video surveillance, biometrics and biomedicine, 25–27 May 2015, Moscow, Russia The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XL-5/W6, 2015 Photogrammetric techniques for video surveillance, biometrics and biomedicine, 25–27 May 2015, Moscow, Russia where O1, O2 = two channels in the opponent color space providing color invariance where O1, O2 = two channels in the opponent color space providing color invariance In common case, at node n the training data Dn are split interactively into left and right subsets using a threshold and the split function according to Equation 8: O3 = the channel in the opponent color space providing intensity invariance    l n r i n l D D D th v F D i D \ ;     (8) (8) The Hue G-SURF (HueG-SURF) is constructed by concatenation of a hue histogram in Hue Saturation Value (HSV) color space with a histogram of G-SURF descriptors. The Hue G-SURF (HueG-SURF) is constructed by concatenation of a hue histogram in Hue Saturation Value (HSV) color space with a histogram of G-SURF descriptors. Such descriptor is scale-invariant and shift-invariant with respect to light intensity due to a Hue histogram. In a Hue histogram, the hue is weighted by the saturation of a pixel and reflected the instabilities in hue. This histogram has 36 dimensions. where Dl, Dr = left and right subsets, respectively th = a threshold F(vi) = a split function vi = ith feature vector where Dl, Dr = left and right subsets, respectively th = a threshold F(vi) = a split function vi = ith feature vector p g p Such descriptor is scale-invariant and shift-invariant with respect to light intensity due to a Hue histogram. In a Hue histogram, the hue is weighted by the saturation of a pixel and reflected the instabilities in hue. This histogram has 36 dimensions. The threshold T is selected randomly in the range T  (min F(vi), max F(vi)). The use of an ensemble of trees trained with small random subsets increases a speed of training and reduces amount of overfitting. Because a random forest dismisses the spatial information of local regions (patches) within a detection window, it produces some false positives, especially when a background has a similar appearance. 5. EXPERIMENTS AND DISCUSSIONS The PASCAL Visual Object Classes (VOC) Dataset and Challenge 20101 was used in training and testing stages. It includes 10,103 and 9,637 images for training and testing, and also 23,374 and 22,992 objects for training and testing, respectively. For experiments, a part of dataset called Action Classification Taster Challenge 2010 was applied. In this part, each person image with 2D pose was annotated by “layouts” of the person with bounding boxes: head, hands, and feets. In total, 322 images containing 439 people were annotated as the training/validation set. The test set involves 441 images with a single person. Figure 2 depicts example images for nine action categories (phoning, walking, running, taking photo, playing instrument, riding bike, riding horse, reading, and using computer) with detected G-SURFs in bounding boxes. Sometimes, a non-significant number of SURFs and G-SURFs can be detected in bounding box due to very small sizes of box, e.g., hand and foot boxes. In these cases, a Kuwahara filter with radius R = 3 was applied in order to receive increased number of feature points. As one can see in Figure 3, where the scaled fragments of images are represented, this additional procedure helps to decide this problem partly. 4.1 Imposed Grid for Poselet Detection However, the experiments show that the rg G-SURF, the OppG- SURF, the HueG-SURF, and the RGBG-SURF provide better results in poselets estimation. 1http://pascallin.ecs.soton.ac.uk/challenges/VOC/ 4.1 Imposed Grid for Poselet Detection The rg histogram is based on the normalized RGB color model, where the components r and g describe the color information by Equation 5 (b is redundant as r + g + b = 1):                                    B G R B B G R G B G R R b g r (5) (5) where R, G, B = Red, Green, Blue components in RGB color space, respectively where R, G, B = Red, Green, Blue components in RGB color space, respectively r, g, b = normalized components where R, G, B = Red, Green, Blue components in RGB color space, respectively r, g, b = normalized components where R, G, B = Red, Green, Blue components in RGB color space, respectively r, g, b = normalized components r, g, b = normalized components Because of normalization, the components r and g are scale- invariant and invariant to lighting changes, shadows and shading (Gevers et al., 2006). This descriptor has 45 dimensions. The Opponent G-SURF (OppG-SURF) analyzes three channels in the opponent color space using G-SURF descriptor (Equation 6): It is mindless to use the predefined aspect ratios as well as the random aspect ratios. It will be reasonable to find Regions Of Interest (ROIs) or pose primitives accurately. If a video sequence is available, then this issue is the solved problem, when the ROIs are detected from the temporal domain in previous frames (Favorskaya, 2012; Favorskaya et al., 2015). Also the hybrid methods based on optical flow and learning of salient regions are possible (e.g., Eweiwi et al. 2015).                                     3 6 2 2 3 2 1 B G R B G R G R O O O (6) (6) If a single still image is available, then one of pixel-based segmentation methods can extract large areas with identical This contribution has been peer-reviewed. oi:10.5194/isprsarchives-XL-5-W6-51-2015 54 This contribution has been peer-reviewed. doi:10.5194/isprsarchives-XL-5-W6-51-2015 54 This contribution has been peer-reviewed. 4.3 Classification Procedure Various classifiers have been applied for object classification such as SVM, Boosting-algorithms, and random forest. The SVM classifier is the well proved technique for general classification. However, SVM is not suitable, when the features have high dimensionality and a set of analysed images is huge. The Boosting-algorithms such as AdaBoost or GentlBoost are the popular machine learning methods but their performance depends critically on the choice of weak classifiers. A random forest is one of the most popular tree-based classification approaches, which is effective in a large variety of high-dimensional tasks such as object detection and object tracking (Ko et al., 2013). The random forest is an ensemble classifier of several randomized decision trees, which have a capacity to analyze big data at high training and runtime speeds. Each tree is grown using some type of randomization. The structure of each tree is binary, and all trees are created in a top- down manner (Breiman, 2001). During the training stage, the random forest uses a random subset from training data initially. 1http://pascallin.ecs.soton.ac.uk/challenges/VOC/ This contribution has been peer-reviewed. doi:10.5194/isprsarchives-XL-5-W6-51-2015 55 The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XL-5/W6, 2015 Photogrammetric techniques for video surveillance, biometrics and biomedicine, 25–27 May 2015, Moscow, Russia Figure 2. The processed person layouts with detected G-SURF Then histogram descriptors had been built in bounding boxes concerning to various body parts. They were classified by random forest technique. During training stage, the separate random forest for heads, hands, and feet were constructed. An example of random forest training (one from a set of trees) is located in Figure 4. Figure 2. The processed person layouts with detected G-SURF Figure 3. Scaled fragments with increased number of G-SURFs 0.870 0.879 Hand 0.936 0.808 Foot Head Head Hand Foot 0.838 Figure 4. Random forest training 0.870 0.870 0.879 Head Head 0.936 Figure 4. Random forest training Except SURF and G-SURF, all four types of color G-SURF were calculated in corresponding color spaces. The experimental results show that the rgG-SURF and the HueG- SURG demonstrated the worst results of recognition and false ratios because they are not robust to light intensity scaling and the G-SURF provided a partial robustness. Table 1 includes the precision evaluation for nine action categories. Except SURF and G-SURF, all four types of color G-SURF were calculated in corresponding color spaces. 4.3 Classification Procedure 45(1), pp. 5- 32. Dalal, N., Triggs, B., 2005. Histograms of oriented gradients for human detection. In: The IEEE Conference on Computer Vision and Pattern Recognition, San Diego, CA, USA, pp. 886-893. Delaitre, V., Laptev, I., Sivic, J., 2010. Recognizing human action in still images: a study of bag-of-features and partial- based representations. In: The British Machine Vision Conference, Aberystwyth, UK, pp. 1-11. Category SURF G- SURF OppG- SURF RGBG- SURF Phoning 28.2 27.3 25.6 25.2 Walking 25.1 24.4 23.1 22.8 Running 28.2 27.1 25.7 25.3 Taking photo 21.8 21.2 20.2 20.1 Playing instrument 49.1 47.9 46.0 45.6 Riding bike 38.9 37.4 36.3 35.8 Riding horse 34.4 33.2 32.5 32.1 Reading 29.8 28.5 27.3 26.9 Using computer 27.2 26.1 25.2 24.8 Table 3. False acceptance rates for nine action categories (%) Eweiwi, A., Cheema, M.S., Bauckhage, C., 2015. Action recognition in still images by learning spatial interest regions from videos. Pattern Recognition Letters, 51, pp. 8-15. Favorskaya, M., 2012. Motion estimation for objects analysis and detection in videos. In: Kountchev, R., Nakamatsu, K. (Eds) Advances in Reasoning-Based Image Processing Intelligent Systems, ISRL vol. 29, Springer-Verlag Berlin Heidelberg, pp. 211-253. Favorskaya, M., Pyankov, D., Popov, A., 2015. Accurate motion estimation based on moment invariants and high order statistics for frames interpolation in stereo vision. In: Tweedale, J.W., Jain, L.C., Watada, J., Howlett, R.J. (Eds) Knowledge- Based Information Systems in Practice, SIST vol. 30, Springer International Publishing Switzerland, pp. 329-351. False rejection rates are higher in comparison to false acceptance rates. It can be explained by usual difficulties in object recognition using still images relative to video-based object recognition in the spatio-temporal domain. Felzenszwalb, P.F., Huttenlocher, D.P., 2005. Pictorial structures for object recognition. Int. J. Comput. Vis., 61(1), pp. 55-79. 4.3 Classification Procedure The experimental results show that the rgG-SURF and the HueG- SURG demonstrated the worst results of recognition and false ratios because they are not robust to light intensity scaling and the G-SURF provided a partial robustness. Table 1 includes the precision evaluation for nine action categories. Category SURF G- SURF OppG- SURF RGBG- SURF Phoning 73.7 75.1 76.8 76.2 Walking 62.2 63.5 64.6 64.1 Running 59.6 60.4 61.3 61.0 Taking photo 77.8 78.1 78.9 78.4 Playing instrument 61.1 62.3 63.1 62.9 Riding bike 58.3 59.7 60.2 59.7 Riding horse 76.6 78.2 78.4 78.2 Reading 68.2 69.7 70.3 69.3 Using computer 83.4 85.1 86.0 85.4 Table 1. Precision results for nine action categories (%) Figure 2. The processed person layouts with detected G-SURF Figure 3. Scaled fragments with increased number of G-SURFs The analysis of received results shows the common tendency: more sizes of bounding boxes provide large number of feature points that leads to better recognition results. Thus, phoning, playing photo, riding horse, and using computer are characterized with better precision results. Tables 2 and 3 contain false rejection rates and false acceptance rates. Also one can see that errors have high values. This may be explained by restriction of current statement of problem. In future, additional procedures, e.g., a graph of body parts, skin detection, skeleton representation, Kinect data analysis, and analysis of surrounding objects, among others, will permit the promising results against current results. The current research shows that the analysis of bounding boxes limits estimators significantly. Figure 3. Scaled fragments with increased number of G-SURFs This contribution has been peer-reviewed. doi:10.5194/isprsarchives-XL-5-W6-51-2015 This contribution has been peer-reviewed. doi:10.5194/isprsarchives-XL-5-W6-51-2015 56 The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XL-5/W6, 2015 Photogrammetric techniques for video surveillance, biometrics and biomedicine, 25–27 May 2015, Moscow, Russia Category SURF G- SURF OppG- SURF RGBG- SURF Phoning 38.9 37.5 36.1 35.9 Walking 40.1 39.2 38.6 38.3 Running 40.2 38.1 37.2 37.1 Taking photo 39.0 38.3 36.8 36.4 Playing instrument 49.1 47.8 45.4 45.1 Riding bike 50.3 49.6 47.9 47.7 Riding horse 51.4 50.2 48.7 48.4 Reading 34.8 33.7 32.5 32.3 Using computer 37.2 36.4 35.2 35.0 Table 2. False rejection rates for nine action categories (%) Bourdev, L., Malik, J., 2009. Poselets: body part detectors trained using 3D human pose annotations. In: The IEEE Conference on Computer Vision, Kyoto, Japan, pp. 1365-1372. Breiman, L., 2001. Random forests. Mach. Learn. 6. CONCLUSION Fidler, S., Dickinson, S., Urtasun, R., 2012. 3D object detection and viewpoint estimation with a deformable 3D cuboid model. In: The Conference on Neural Information Processing Systems, Lake Tahoe, Nevada, USA, pp .620-628. In this study, the poselets estimation is proposed for recognition of human active actions. Our approach deals with accurate segmentation of body parts in a still image by a possibility of the temporal data extraction from a video sequence or pixel- based segmentation method in order to receive the images of body parts. The experiments with G-SURF lead to the descriptor invariant to color/lighting conditions. The SPOD is based on color G-SURF family. Better results were received using OppG-SURF and RGBG-SURF. For classification of common SPOD, a random forest classification was used as a fast and effective procedure during work with big data representing various poselets of various human active actions. PASCAL VOC Dataset and Challenge 2010 provided the test material for the training and the testing stages. The precision for nine action categories such as phoning, walking, running, taking photo, playing instrument, riding bike, riding horse, reading, and using computer achieves on the average of 61-68% for all 3D poses locations and 82-86% for front poses locations regarding to action categories. Gevers, T., van de Weijer, J., Stokman, H., 2006. Color feature detection: an overview. In Lukac, R., Plataniotis, K.N. (Eds) Color image processing: methods and applications. University of Toronto, Ontario, Canada: CRC press, pp. 203-226. Glasner, D., Galun, M., Alpert, S., Basri, R., Shakhnarovich, G., 2011. 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This contribution has been peer-reviewed. doi:10.5194/isprsarchives-XL-5-W6-51-2015 This contribution has been peer-reviewed. doi:10.5194/isprsarchives-XL-5-W6-51-2015 This contribution has been peer-reviewed. doi:10.5194/isprsarchives-XL-5-W6-51-2015 57 57 The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XL-5/W6, 2015 Photogrammetric techniques for video surveillance, biometrics and biomedicine, 25–27 May 2015, Moscow, Russia Yang, W., Wang, Y., Mori, G., 2010. Recognizing human actions from still images with latent poses. In: The IEEE Conference on Computer Vision and Pattern Recognition, San Francisco, USA, pp. 2030-2037. Khan, F.S., Anwer, R.M., van de Weijer, J., Bagdanov, A.D., Lopez A.M., Felsberg M. 2013. Coloring Action Recognition in Still Images. Int. J. of Computer Vision, 105(3), pp. 205-221. Ko, B.C., Kwak, J.Y., Nam, J.Y., 2013. 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https://openalex.org/W1995072592	https://europepmc.org/articles/pmc2964299?pdf=render	English	null	Unforeseen Costs of Cutting Mosquito Surveillance Budgets	PLoS neglected tropical diseases	2,010	cc-by	3,758	Abstract Competing Interests: The authors have declared that no competing interests exist. * E-mail: gmvazqu@emory.edu Unforeseen Costs of Cutting Mosquito Surveillance Budgets Gonzalo M. Vazquez-Prokopec1*, Luis F. Chaves1,2, Scott A. Ritchie3,4, Joe Davis3, Uriel Kitron1 1 Department of Environmental Studies, Emory University, Atlanta, Georgia, United States of America, 2 Programa de Investigacio´n en Enfermedades Tropicales, Escuela de Medicina Veterinaria, Universidad Nacional, Heredia, Costa Rica, 3 Cairns Tropical Public Health Unit, Queensland Health, Cairns, Queensland, Australia, 4 School of Public Health, Tropical Medicine and Rehabilitation Sciences, James Cook University, Cairns, Queensland, Australia Abstract A budget proposal to stop the U.S. Centers for Disease Control and Prevention (CDC) funding in surveillance and research for mosquito-borne diseases such as dengue and West Nile virus has the potential to leave the country ill- prepared to handle new emerging diseases and manage existing ones. In order to demonstrate the consequences of such a measure, if implemented, we evaluated the impact of delayed control responses to dengue epidemics (a likely scenario emerging from the proposed CDC budget cut) in an economically developed urban environment. We used a mathematical model to generate hypothetical scenarios of delayed response to a dengue introduction (a consequence of halted mosquito surveillance) in the City of Cairns, Queensland, Australia. We then coupled the results of such a model with mosquito surveillance and case management costs to estimate the cumulative costs of each response scenario. Our study shows that halting mosquito surveillance can increase the management costs of epidemics by up to an order of magnitude in comparison to a strategy with sustained surveillance and early case detection. Our analysis shows that the total costs of preparedness through surveillance are far lower than the ones needed to respond to the introduction of vector-borne pathogens, even without consideration of the cost in human lives and well-being. More specifically, our findings provide a science-based justification for the re-assessment of the current proposal to slash the budget of the CDC vector-borne diseases program, and emphasize the need for improved and sustainable systems for vector-borne disease surveillance. Citation: Vazquez-Prokopec GM, Chaves LF, Ritchie SA, Davis J, Kitron U (2010) Unforeseen Costs of Cutting Mosquito Surveillance Budgets. PLoS Negl Trop Dis 4(10): e858. doi:10.1371/journal.pntd.0000858 Editor: Ann M. Powers, Centers for Disease Control and Prevention, United States of America Editor: Ann M. Powers, Centers for Disease Control and Prevention, United States of America Received July 28, 2010; Accepted September 28, 2010; Published October 26, 2010 Received July 28, 2010; Accepted September 28, 2010; Published October 26, 2010 Copyright: 2010 Vazquez-Prokopec 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 Emory University (Department of Environmental Studies internal support). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. October 2010 \| Volume 4 \| Issue 10 \| e858 Author Summary A strong network of state and local health departments rely on CDC funds for personnel and routine seasonal testing of mosquitoes for WNV and other viruses. Indeed, one of the reasons the recent emergence of dengue in mainland US (after a 50-year hiatus [12]) was rapidly detected and contained is the through the presence in Florida of the CDC- supported vector surveillance network. (res = 2, 4, 6 and 8 weeks) were computed by estimating the number of cases in the absence of control (between t0 and res) using R0, and then generating the rest of each epidemic time series by multiplying the number of cases by the estimated post intervention Rt in the original series. A response within 2 weeks of an introduction was assumed to occur only when active vector surveillance was in place (incurring a continuous cost), whereas delays in response of 6–8 weeks occurred when active disease and vector surveillance were eliminated. Direct and indirect costs per case provided by the Cairns Public Health Unit of Queensland Health, Australia (Table 1), were used to estimate the cumulative cost (in 2009 US$) of each hypothetical response scenario (Figure 1 C–D). Costs were transformed from AU$ to US$ using each year’s average exchange rate, and from 2003 US$ to 2009 US$ values using the Gross Domestic Product (GDP) deflator [6]. Direct costs included vector control, case diagnosis and blood bank screening costs, whereas indirect costs included work days lost due to disease. Our analysis assumed absence of dengue hemorrhagic fever manifestations (consequence of the lack of co-circulation of other dengue virus strains in epidemic settings) and no costs associated with subclinical infections. Without CDC funds, mosquito testing would be halted, and detection of transmission events or novel viral introductions significantly delayed (with response delayed by even more than 8 weeks), turning CDC into a reactive rather than preventive health service. Our analysis clearly shows that the total costs of preparedness through surveillance are far lower than the ones needed to respond to the introduction of vector-borne pathogens, even without consideration of the cost in human lives and well-being. Our economic analysis provides strong ammunition from an ethical, economic and scientific standpoint for lawmakers to retain the investments in this cost-effective preventive public health strategy. In fact, our analysis points to the need for more, rather than less, funding for vector-borne disease surveillance. Introduction The 2011 U.S. fiscal year proposed budget cut of $26.7 million from the CDC vector-borne diseases program [1] could virtually paralyze surveillance and research activities directed at diseases already circulating in the U.S such as dengue and West Nile virus (WNV), and jeopardize the capability of the existing health infrastructure for early detection of other exotic mosquito-transmitted pathogens such as Rift Valley fever, Japanese encephalitis and chikungunya virus [1]. Surveillance is the first line of defense against infectious diseases [2], guides health agencies’ response to infectious threats, optimizes resources by focusing interventions on target areas, and generates invaluable information for health providers and policy makers [2]. We present here a case study where we couple a mathematical model with cost analysis to evaluate the economic impact of different response scenarios to the introduction of a vector-transmitted pathogen of public health importance into an economically developed urban environment. Data from two well-documented and successfully controlled dengue fever outbreaks introduced by viremic travelers into the city of Cairns, Queensland, Australia in 2003 and 2009 [3,4] were used to derive the basic reproductive number (R0) and the effective reproduction number (Rt) of dengue transmission. R0 represent the average number of secondary cases after the introduction of an infection, and was estimated by fitting an exponential function to the observed weekly epidemic curves before vector control interventions began (6 weeks in 2003 and 4 weeks in 2009, Figure 1 A–B) following the method of Nishiura et al. [5]. Rt represent the average number of secondary cases per primary case at time t of each outbreak and was estimated by accumulating the number of cases in biweekly periods (the average generation time of dengue is ,14 days) and computing the ratio between consecutive two-week periods. Hypothetical epidemic curves for the 2003 (Figure 1A) and 2009 (Figure 1B) outbreaks under different scenarios for response times (res) of vector control activities to a dengue introduction ww October 2010 \| Volume 4 \| Issue 10 \| e858 1 www.plosntds.org www.plosntds.org The Cost of Cutting Mosquito Budgets 2003 and 2009 epidemics, respectively (Figure 1). Responding to the same outbreaks 4–6 weeks later (res = 6–8) would have resulted in cumulative costs of containing the 2003 and 2009 outbreaks that are 86 (or US$ 13 million; Figure 1 C) and 346 (or US$382 million; Figure 1 D) times as high, respectively, than a strategy based on ongoing active surveillance. Author Summary Surveillance has served as a basis for important public health responses to new threats, and as a source of invaluable information for health providers and policy makers. A budget proposal to stop the U.S. Centers for Disease Control and Prevention (CDC) funding in surveillance and research for mosquito-borne diseases such as dengue and West Nile virus has the potential to leave the country ill-prepared to handle new emerging diseases and manage existing ones. The present article uniquely integrates infectious disease models with economic analysis, taking advantage of a unique detailed dataset. By coupling a mathematical model with cost analysis we were able to evaluate the impact of delayed control responses to dengue fever, a mosquito-transmit- ted disease of global importance, in an economically developed urban environment. Our analysis clearly shows that the total costs of preparedness through surveillance are far lower than the ones that follow the introduction of vector-borne pathogens. Our findings will help provide a science-based justification for re- assessment of the current proposal to slash the budget of the CDC vector-borne diseases program. More generally our study demonstrates the power of rigorous analysis of carefully collected data for a balanced assessment of the economic implications of a public health program shift. Without a strong human and vector surveillance system, detection and response to emerging vector-borne diseases that can present, in many instances, undetermined symptoms in humans could be severely impaired. The emergence of WNV in New York City in 1999 is a clear example of the consequences that a delayed response can have on the outcome of a novel arboviral introduction [8,9]. The first glimpse of WNV transmission occurred with the notification of unusual bird deaths in late June. The incorrect diagnosis of a cluster of human cases as St. Luis encephalitis in late August prompted the initiation of vector control actions, almost 2 months since the detection of bird deaths [8,9]. By the time vector control was in place and WNV confirmed as the putative source of human and bird infections, the infection could not be contained (particularly in the bird population), and subsequently progressed throughout the US, generating 28,961 WNV human cases and 1,130 fatalities by the end of 2008 [10]. Vector-borne disease surveillance in the U.S. improved significantly after this failure to contain WNV [11]. Author Summary The probability for early detection of an introduction of a vector borne disease agent, or for rapid interruption of transmission if an outbreak were to occur, are a direct function of adequate funding for vector borne disease research and surveillance. Introduction By the 9th week of an outbreak the costs accrued in controlling it increased exponentially and far surpassed the costs of a strategy with sustained surveillance and early case detection (res = 2) (Figure 1 C–D). Thus, a delayed reaction to both Cairns dengue outbreaks would have resulted in drastically escalated total costs of up to US$ 382 million. Indeed, a slight difference in the virulence of the invading strain (DR0 = 0.1 between outbreaks) would have increased total costs by one order of magnitude (Figure 1 C–D). Notably, our predictions show that the costs to contain the 2009 outbreak in a city with a climate comparable to Miami, but with ,10% of the population, would have been an order of magnitude higher than the proposed CDC budget cut that will impact the whole US. www.plosntds.org www.plosntds.org Results and Discussion The dengue outbreaks in Cairns demonstrate the vulnerability of developed countries to mosquito-borne pathogens that are major international public health concerns [7]. Our analysis shows that delaying control responses translates into an exponential increase in both the number of human cases and health costs (Figure 1). The cumulative cost of a strategy with active surveillance and res of 2 was US$ 0.15 and US$1.1 million for October 2010 \| Volume 4 \| Issue 10 \| e858 2 The Cost of Cutting Mosquito Budgets Figure 1. Impacts of hypothetical scenarios of delayed response of vector control to Dengue virus outbreaks. The basic reproduct number, R0 (the average number of secondary cases after the introduction of an infection) for the 2003 and 2009 dengue fever outbreaks t affected the city of Cairns, Australia, was estimated by fitting an exponential function to the observed weekly epidemic curves before vector con interventions began (6 weeks in 2003 and 4 weeks in 2009). The effective reproduction number, Rt (the average number of secondary cases primary case at time t) of each outbreak was estimated by accumulating the number of cases in biweekly periods (the average generation time dengue is ,14 days) and computing the ratio between consecutive two-week periods. The hypothetical epidemic curves for the 2003 (A) and 2 (B) outbreaks under different scenarios for response times (res) of vector control activities to a dengue introduction (res = 2, 4, 6 and 8 weeks) w computed by estimating the number of cases in the absence of control (between t0 and res) using R0, and then generating the rest of each epide time series by multiplying the number of cases by the estimated post intervention Rt in the original series. Blue lines indicate a faster response t than in the actual outbreak, red lines indicate scenarios where the response is delayed in comparison to the actual outbreak, and green lines indic the actual outbreak. Values on top of the green lines are estimates for Rt. Cumulative cost (in 2009 US$) of each res scenario were estimated for 2003 (C) and 2009 (D) outbreaks. Figure legends refer to each res scenario (A,B) and to the final epidemic size of each scenario (C,D). doi:10 1371/journal pntd 0000858 g001 The Cost of Cutting Mosquito Budg Figure 1. Impacts of hypothetical scenarios of delayed response of vector control to Dengue virus outbreaks. Results and Discussion The basic reproduct number, R0 (the average number of secondary cases after the introduction of an infection) for the 2003 and 2009 dengue fever outbreaks t affected the city of Cairns, Australia, was estimated by fitting an exponential function to the observed weekly epidemic curves before vector cont interventions began (6 weeks in 2003 and 4 weeks in 2009). The effective reproduction number, Rt (the average number of secondary cases primary case at time t) of each outbreak was estimated by accumulating the number of cases in biweekly periods (the average generation time Figure 1. Impacts of hypothetical scenarios of delayed response of vector control to Dengue virus outbreaks. The basic reproduction number, R0 (the average number of secondary cases after the introduction of an infection) for the 2003 and 2009 dengue fever outbreaks that affected the city of Cairns, Australia, was estimated by fitting an exponential function to the observed weekly epidemic curves before vector control interventions began (6 weeks in 2003 and 4 weeks in 2009). The effective reproduction number, Rt (the average number of secondary cases per primary case at time t) of each outbreak was estimated by accumulating the number of cases in biweekly periods (the average generation time of dengue is ,14 days) and computing the ratio between consecutive two-week periods. The hypothetical epidemic curves for the 2003 (A) and 2009 (B) outbreaks under different scenarios for response times (res) of vector control activities to a dengue introduction (res = 2, 4, 6 and 8 weeks) were computed by estimating the number of cases in the absence of control (between t0 and res) using R0, and then generating the rest of each epidemic time series by multiplying the number of cases by the estimated post intervention Rt in the original series. Blue lines indicate a faster response time than in the actual outbreak, red lines indicate scenarios where the response is delayed in comparison to the actual outbreak, and green lines indicate the actual outbreak. Values on top of the green lines are estimates for Rt. Cumulative cost (in 2009 US$) of each res scenario were estimated for the 2003 (C) and 2009 (D) outbreaks. Figure legends refer to each res scenario (A,B) and to the final epidemic size of each scenario (C,D). doi:10.1371/journal.pntd.0000858.g001 October 2010 \| Volume 4 \| Issue 10 \| e858 3 www.plosntds.org The Cost of Cutting Mosquito Budgets Table 1. Author Contributions Conceived and designed the experiments: GMVP LFC SAR UK. Performed the experiments: GMVP LFC. Analyzed the data: GMVP LFC. Contributed reagents/materials/analysis tools: SAR JD. Wrote the paper: GMVP LFC SAR UK. We thank Helen Faddy from the Australian Red Cross Blood Service for providing data on costs of blood-bank screening. 12. Centers for Disease Control and Prevention (2010) Locally acquired Dengue– Key West, Florida, 2009–2010. MMWR Morb Mortal Wkly Rep 59: 577–581. 11. Centers for Disease Control and Prevention (2006) Assessing capacity for surveillance, prevention, and control of West Nile virus infection–United States, 1999 and 2004. MMWR Morb Mortal Wkly Rep 55: 150–153. Results and Discussion 4Information provided by the Australian Red Cross Blood Service. 1DART’s responsibility is to implement mosquito prevention and control. In large outbreaks DART is supplemented by environmental health and municipal agents. 2Vector control encompasses selective indoor insecticide residual spraying (SC 2.5% lambda-cyhalothrin, Demand) and larval control/source reduction activities (removal of small containers and treatment of large containers with S-methophene pellets or residual surface sprays) in premises within 100 meters of a case. 3Serum samples are forwarded to the reference laboratory where they are screened for the presence of anti-dengue IgM and IgG using a combined pool of flavivirus antigens in capture ELISA assays. Positive IgM samples are further analyzed using flavivirus-specific IgM ELISA capture assays in order to identify the serotype of the infecting dengue virus. Additionally, real-time TaqMan reverse transcriptase-polymerase chain reaction is performed to detect dengue virus RNA. 4Information provided by the Australian Red Cross Blood Service. to loose, on average, 5 work days. Daily costs were estimated by dividing the median monthly income in Cairns (US$ 25,419; source stics) by the number of working days (250). y 5Each cased was assumed to loose, on average, 5 work days. Daily costs were estimated by dividing the median monthly income in Australian Bureau of Statistics) by the number of working days (250). 10. Lindsey NP, Staples JE, Lehman JA, Fischer M (2010) Surveillance for human West Nile virus disease - United States, 1999–2008 MMWR Surveill Summ. 59: 1–17. 7. Gubler DJ, Kuno G (1997) Dengue and dengue hemorrhagic fever. Gubler DJ, Kuno G, eds. London, UK: CAB International; 1997. Results and Discussion Cost estimates per month (during surveillance) and per case (during an outbreak) to prevent and control dengue fever introductions in Cairns, Australia. Item Cost units Cost (2009 U$) Surveillance Dengue action response team (DART)1 per month 25,967 Control2 Personnel per case 1,336 Travel per case 282 Vehicle use per case 64 Insecticides per case 279 Miscellaneous expenses per case 177 Diagnosis Diagnosis tests3 per case 96 Blood bank screening4 per case 805 Days lost due to disease5 per case 508 1DART’s responsibility is to implement mosquito prevention and control. In large outbreaks DART is supplemented by environmental health and municipal agents. 2Vector control encompasses selective indoor insecticide residual spraying (SC 2.5% lambda-cyhalothrin, Demand) and larval control/source reduction activities (removal of small containers and treatment of large containers with S-methophene pellets or residual surface sprays) in premises within 100 meters of a case. 3Serum samples are forwarded to the reference laboratory where they are screened for the presence of anti-dengue IgM and IgG using a combined pool of flavivirus antigens in capture ELISA assays. Positive IgM samples are further analyzed using flavivirus-specific IgM ELISA capture assays in order to identify the serotype of the infecting dengue virus. Additionally, real-time TaqMan reverse transcriptase-polymerase chain reaction is performed to detect dengue virus RNA. 4Information provided by the Australian Red Cross Blood Service. 5 Table 1. Cost estimates per month (during surveillance) and per case (during an outbreak) to prevent and control dengue fever introductions in Cairns, Australia. Table 1. Cost estimates per month (during surveillance) and per case (during an outbreak) to prevent and control dengue fever introductions in Cairns, Australia. 1DART’s responsibility is to implement mosquito prevention and control. In large outbreaks DART is supplemented by environmental health and municipal agents. 2Vector control encompasses selective indoor insecticide residual spraying (SC 2.5% lambda-cyhalothrin, Demand) and larval control/source reduction activities (removal of small containers and treatment of large containers with S-methophene pellets or residual surface sprays) in premises within 100 meters of a case. 3Serum samples are forwarded to the reference laboratory where they are screened for the presence of anti-dengue IgM and IgG using a combined pool of flavivirus antigens in capture ELISA assays. Positive IgM samples are further analyzed using flavivirus-specific IgM ELISA capture assays in order to identify the serotype of the infecting dengue virus. Additionally, real-time TaqMan reverse transcriptase-polymerase chain reaction is performed to detect dengue virus RNA. References 8. United States General Accounting Office (2000) Report to Congressional Requesters. West Nile Virus Outbreak – Lessons for public health preparedness. Available at [http://www.gao.gov/archive/2000/he00180.pdf]. 8. United States General Accounting Office (2000) Report to Congressional Requesters. West Nile Virus Outbreak – Lessons for public health preparedness. Available at [http://www.gao.gov/archive/2000/he00180.pdf]. 1. Couzin-Frankel J (2010) Infectious diseases. Fears of lax surveillance if CDC program cut. Science 328: 1088. 2. Berkelman RL, Bryan RT, Osterholm MT, LeDuc JW, Hughes JM (1994) Infectious disease surveillance: a crumbling foundation. Science 264: 368–370. 9. Centers for Disease Control and Prevention (1999) Outbreak of West Nile-like viral encephalitis–New York, 1999. MMWR Morb Mortal Wkly Rep 48: 845–849. 3. Hanna JN, Ritchie SA (2009) Outbreaks of dengue in north Queensland. Commun Dis Intell 33: 32–33. 4. Hanna JN, Ritchie SA, Richards AR, Taylor CT, Pyke AT, et al. (2006) Multiple outbreaks of dengue serotype 2 in north Queensland, 2003/04. Aust N Z J Public Health 30: 220–225. 4. Hanna JN, Ritchie SA, Richards AR, Taylor CT, Pyke AT, et al. (2006) Multiple outbreaks of dengue serotype 2 in north Queensland, 2003/04. Aust N Z J Public Health 30: 220–225. 10. Lindsey NP, Staples JE, Lehman JA, Fischer M (2010) Surveillance for human West Nile virus disease - United States, 1999–2008 MMWR Surveill Summ. 59: 1–17. 5. Nishiura H, Chowell G, Heesterbeek H, Wallinga J (2009) The ideal reporting interval for an epidemic to objectively interpret the epidemiological time course. J R Soc Interface 7: 297. 5. Nishiura H, Chowell G, Heesterbeek H, Wallinga J (2009) The ideal reporting interval for an epidemic to objectively interpret the epidemiological time course. J R Soc Interface 7: 297. 6. U.S. Department of Commerce: Bureau of Economic Analysis [http://www. bea.gov/]. 6. U.S. Department of Commerce: Bureau of Economic Analysis [http://www. bea.gov/]. 7. Gubler DJ, Kuno G (1997) Dengue and dengue hemorrhagic fever. Gubler DJ, Kuno G, eds. London, UK: CAB International; 1997. www.plosntds.org October 2010 \| Volume 4 \| Issue 10 \| e858 4 www.plosntds.org
https://openalex.org/W3041661131	https://www.imrpress.com/journal/CEOG/46/6/10.12891/ceog4757.2019/pdf	English	null	Prenatal diagnosis of fetal gallbladder duplication associated with uncommon chromosomal anomaly (46, XX, t(X;10) (p11.2;q24.3) [20])	Clinical and experimental obstetrics & gynecology	2,019	cc-by	1,368	Introduction female was delivered via cesarean section for primiparous breech presentation. Antenatal care was only remarkable for the diagnosis of small for gestational age fetus (without and abnormal fetal well being problems and abnormal prenatal doppler findings). Neona- tal abdominal sonography on postnatal day 3 confirmed the pre- natal diagnosis of gallbladder duplication (two gallbladders with two cystic canal opening to one common bile duct). The baby was discharged at day 7 without any complications. No additional ul- trasound abnormalities either prenatal or postnatal screening were reported. The woman also had the same chromosomal anomaly and abdominal sonography of woman revealed the diagnosis of cystic canal duplication (one gallbladder with two cystic canal opening to one common bile duct). Fetal gallbladder duplication is a rare congenital malfor- mation, occurring in about one in 4,000 births, resulting in two separate gallbladder cavities, each with a cystic duct. Gallbladder anomalies, including cholelithiasis, chole- dochal cysts, and agenesis, have been observed in utero [1, 2]. In the literature only a few cases of fetal gallbladder du- plication in utero (only four cases) were reported. All re- ported cases were not associated with additional congenital or genetic anomalies. A novel chromosomal mutation (46, XX, t(X;10) (p11.2;q24.3) [20]) with duplicated fetal gall- bladder which has not been reported previously was re- ported. Figure 1. — (a) Axial section of fetal abdomen on sonography shows two hypoechoic cyst-like structures (arrows, fetal gallblad- der duplication). (b) Coronal T2 weighed MRI shows two rectan- gular cyst-like structure located at the lower border of the liver (arrows, fetal gallbladder duplication) at 26 weeks’ of pregnancy. Prenatal diagnosis of fetal gallbladder duplication associated with uncommon chromosomal anomaly (46, XX, t(X;10) (p11.2;q24.3) [20]) E.H. Comert1, H. Sal1, C. Kart1, E.S. Guvendag Guven1, S. Guven1 1Department of Obstetrics and Gynecology, Karadeniz Technical University, School of Medicine, Trabzon (Turkey) Summary Summary Fetal gallbladder duplication is a rare congenital malformation. In the literature only a few cases of fetal gallbladder duplication in utero is reported. A 22-year-old woman was referred to this hospital at 26 weeks of gestation for a routine ultrasound examination. A fetal duplication of gallbladder accompanied was diagnosed with ultrasonography (axial section of fetal abdomen on sonography showed two hypoechoic cyst-like structures). The diagnosis of duplication of gallbladder, was also confirmed with MRI. The result of cordo- centesis was reported as 46, XY, t(X;10) (p11.2;q24.3) [20]. Antenatal/natal/postnatal care was unremarkable. A novel chromosomal mutation (46, XX, t(X;10) (p11.2;q24.3) [20]) with duplicated fetal gallbladder which has not been reported previously was described. Key words: Chromosomal anomaly; Fetal gallbladder duplication; Ultrasonography; MRI. Revised manuscript accepted for publication May 22, 2018 Clin. Exp. Obstet. Gynecol. - ISSN: 0390-6663 XLVI, n. 6, 2019 doi: 10.12891/ceog4757.2019 CEOG Clinical and Experimental Obstetrics & Gynecology CEOG Clinical and Experimental Obstetrics & Gynecology Case Report E.H. Comert1, H. Sal1, C. Kart1, E.S. Guvendag Guven1, S. Guven1 1Department of Obstetrics and Gynecology, Karadeniz Technical University, School of Medicine, Trabzon (Turkey) Case Report A 22-year-old woman (gravidity 1, parity 0) was referred to the present hospital at 26 weeks of gestation for a routine ultrasound examination. Obstetric and medical histories were all unremark- able. A fetal duplication of gallbladder accompanied was diag- nosed with ultrasonography (Figure 1a). The diagnosis of duplication of gallbladder (Figure 1b), was also confirmed with MRI. In order to rule out fetal aneuploidy, cordocentesis was sug- gested. The result of cordocentesis was reported as 46, XY, t(X;10) (p11.2;q24.3) [20]. C-banding with trypsin-Giemsa and high resolution banding techniques were used to assess this anomaly. The patient was counseled about the implication of this chromosomal anomaly based on literature findings (infertility and gallbladder diseases). Fetal sonographic follow-up was also con- firmed the initial diagnosis.At 39 weeks of gestation a 2,480-gram Figure 1. — (a) Axial section of fetal abdomen on sonography shows two hypoechoic cyst-like structures (arrows, fetal gallblad- der duplication). (b) Coronal T2 weighed MRI shows two rectan- gular cyst-like structure located at the lower border of the liver (arrows, fetal gallbladder duplication) at 26 weeks’ of pregnancy. Revised manuscript accepted for publication May 22, 2018 7847050 Canada Inc. www.irog.net Clin. Exp. Obstet. Gynecol. - ISSN: 0390-6663 XLVI, n. 6, 2019 doi: 10.12891/ceog4757.2019 7847050 Canada Inc. www.irog.net Published:10 December 2019 E.H. Comert, H. Sal, C. Kart, E.S. Guvendag Guven, S. Guven 981 Discussion of such chromosomal anomaly (infertility, gallbladder dis- eases). Antenatal care should include the risks of small fetus and need of cesarean delivery. The diagnosis of gallbladder duplication is very rarely carried out during routine obstetric sonography. The gall- bladder appears as a small, hypoechoic, oval or rectangular cyst-like structure located at the lower border of the liver, close to the intestinal loops, and to the right of the intra- hepatic umbilical vein [2]. The fetal gallbladder is visual- ized in 65 -82% of fetuses at 24-27 weeks’ gestation [3]. Although by using a transvaginal approach at 14-16 weeks’ gestation the detection rate may be increased to more than 99% [2]. In this case, MRI was planned when duplication of gall- bladder was diagnosed on routine prenatal obstetric ultra- sonography. The diagnosis was confirmed with MRI. Fetal karyotype analysis revealed 46, XY, t(X;10) (p11.2;q24.3) [20]. Literature review failed to reveal the association of such chromosomal anomaly and fetal gallbladder duplica- tion. When clinicians suspect fetal gallbladder anomaly, MRI for confirming the diagnosis is suggested and fetal karyotype analysis to discover such unreported chromoso- mal anomaly. Gallbladder duplication should be considered when an additional cystic structure is seen in the right upper quad- rant. Its specific location in the gallbladder fossa and its characteristic appearance are helpful in distinguishing it from other pathologic entities, particularly choledochal and duodenal duplication cysts, both of which occur in this lo- cation. A structure lying parallel to the gallbladder was not identified in neither of these conditions. In particular, du- plication cyst of the duodenum is contiguous with the de- scending duodenum and appears as an anechoic mass with strong back wall echoes. This may be associated with var- ious congenital anomalies [4]. References [1] Suchet I.B., Labatte M.F., Dyck C.S., Salgado L.A.: “Fetal cholelithi- asis: a case report and review of the literature”. J. Clin. Ultrasound, 1993, 21, 198. [2] Blazer S., Zimmer E.Z., Bronshtein M.: “Nonvisualization of the fetal gallbladder in early pregnancy: comparison with clinical out- come”. Radiology, 2002, 224, 379. [3] Hata K., Aoki S., Hata T., Murao F., Kitao M.: “Ultrasonographic identification of the human fetal gallbladder in utero”. Gynecol. Ob- stet. Invest., 1987, 23, 79. [4] Narlawar R.S., Rao J.R., Karmarkar S.J., Gupta A., Hira P.: “Sono- graphic findings in a duodenal duplication cyst”. J. Clin. Ultrasound, 2002, 30, 566. The differential diagnosis of gallbladder duplication from some gallbladder diseases is often difficult in woman or man. It is often found incidentally during the surgery for cholelithiasis [5-7]. The diagnosis duplication of gallblad- der in utero may not be easily done since it does not have specific symptoms, only strict sonographic anatomic survey may give the opportunity to make the diagnosis of such rare anomaly. There have been no reported genetic or biochem- ical marker for such anomaly. [5] Horattas M.C.: “Gallbladder duplication and laparoscopic manage- ment”. J. Laparoendosc. Adv. Surg. Tech. A., 1998, 8, 231. [6] Ozgen A., Akata D., Arat A., Demirkazik F.B., Ozmen M.N., Akhan O.: “Gallbladder duplication: imaging findings and differential con- siderations”. Abdom. Imaging, 1999, 24, 285. [7] Shirahane K., Yamaguchi K., Ogawa T., Shimizu S., Yokohata K., Mizumoto K., Tanaka M.: “Gallbladder duplication successfully re- moved laparoscopically using endoscopic nasobiliary tube”. Surg. Endosc., 2003, 17, 1156. In case of suspicious fetal gallbladder anomaly in preg- nancy, fetal sonography reveals two similar cystic struc- tures located in the right upper quadrant, and the woman should be subjected to MRI study to confirm the diagnosis of gallbladder duplication. Following confirmation the di- agnosis, fetal and/or parent karyotype analyses should be advised to exclude de novo or inherited genetic anomalies. Patients should be counseled about the clinical implication Corresponding Author: S. GUVEN, M.D. Department of Obstetrics and Gynecology School of Medicine, Karadeniz Technical University 61080 Trabzon (Turkey) e-mail: drsuleymanguven@yahoo.com Corresponding Author: S. GUVEN, M.D. Department of Obstetrics and Gynecology School of Medicine, Karadeniz Technical University 61080 Trabzon (Turkey) e-mail: drsuleymanguven@yahoo.com
https://openalex.org/W4319438026	https://www.researchsquare.com/article/rs-1160643/latest.pdf	English	null	Perceived barriers, benefits, facilitators, and attitudes of health professionals towards type 2 diabetes management in Oujda, Morocco: a qualitative focus group study	International journal for equity in health	2,023	cc-by	5,541	Perceived Barriers, Benefits, Facilitators, and Attitudes of Health Professionals Towards Type 2 Diabetes Management in Oujda, Morocco: A Qualitative Focus Group Study Usman Sani Dankoly University of Antwerp Dirk Vissers (  dirk.vissers@uantwerpen.be ) University of Antwerp Souad Ben El Mostafa University Mohammed Premier Abderrahim Ziyyat University Mohammed Premier Bart Van Rompaey University of Antwerp Paul Van Royen University of Antwerp Abdellatif Maamri University Mohammed Premier Research Article License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Additional Declarations: No competing interests reported. Additional Declarations: No competing interests reported. Version of Record: A version of this preprint was published at International Journal for Equity in Health on February 7th, 2023. See the published version at https://doi.org/10.1186/s12939-023-01826-5. Page 1/14 Page 1/14 Page 1/14 Abstract Background: In Morocco, the treatment of type 2 diabetes (T2D) is mainly focused on medication and only 2% of patients are coached towards a healthier lifestyle. In Oujda, Eastern Morocco the prevalence of T2D is 10.2%, and the current trend is alarming, especially for women. Therefore, the aim of this study is to explore healthcare professionals' (HCP) views about the perceived barriers and benefits towards an integrated care approach in primary healthcare centers (PHCCs) to T2D management in Oujda. Methods: A descriptive study using focus groups in 8 PHCCs. This resulted in a sample of 5 doctors and 25 nurses caring for diabetes patients. The transcripts of all conversations were coded to allow for thematic analysis. Results: The participants mentioned different barriers to an integrated approach of DM management:: excessive workload; poor reimbursement policy; lack of staff and equipment; interrupted drug supply; poor working environment; limited referral; gap in the knowledge of general practitioners; health beliefs; poverty; advanced age; gender; the use of psychotropic drugs. An integrated approach could be facilitated by simplified electronic records and referral; uninterrupted free care; staff recruitment; continuous professional development; internships. Benefits: structured care; promotion of care in PHCCs; empowerment of self-management. Conclusion: HCP views reflect the urge to strengthen the management of T2D in PHCCs. There is a need for HCP with expertise in physical activity and nutrition to solve the current gap in the multidisciplinary integrated care approach. The specific local context can contribute to patients' reluctance to change their lifestyles and is a challenge to provide care in an efficient and sustainable manner. More research is needed to see how a patient-centered multidisciplinary approach to T2D management can help motivate patients in Morocco to change to a healthier lifestyle. Settings This study was carried out across 8 PHCCs located in both urban and rural regions in Oujda. T2D management in these settings is currently limited to GPs and nurses. There were no specialist diabetes nurses; instead, the nurses who are responsible for the provision of care for patients with diabetes are called general nurses. The interview took place between November 2019 and February 2020. Most nurses are between 25 and 55 years old while GPs are between 40 and 55 years old. Each focus group took place either in the doctor’s or medical director’s office. Design We used a qualitative exploratory research design. Focus groups were used to collect data, as this is an effective way to use group interactions to explore perceptions of HCPs about T2D management [10]. Participants A purposive sample of 30 healthcare professionals (5 doctors and 25 nurses) participated in a focus group interview (Table 1). Table 1: Distribution of HCPs in PHCCs for focus group Table 1: Distribution of HCPs in PHCCs for focus group Introduction In 2015, worldwide around 415 million people have diabetes and by 2040, this figure is predicted to rise to 642 million people [1]. Type 2 diabetes (T2D) accounts for 90% of the total diabetes burden [2]. In Morocco, as in many Middle Eastern and North African countries, T2D prevalence is increasing at unprecedented rates more than in any part of the world [3]. Physical inactivity and unhealthy dietary habits have contributed to a high prevalence of T2D, up to 10.6 % among adults Moroccans between 2017 to 2018 [4]. In Oujda, Eastern Morocco the prevalence of T2D is 10.2% [5] and the current trend is alarming, especially for women [6]. An international diabetes study shows that Moroccan healthcare professionals (HCPs) mainly focus on the monitoring and medical treatment of diabetes. Only 2% of the study population received lifestyle recommendations [7]. According to 2018 World Bank reports, only 6.35 million people (19%) percent of the population are covered by subsidized basic health insurance. Private hospitals are only accessible by money whilst quality of care remains a challenge for public primary healthcare centers (PHCCs) [8]. Page 2/14 Page 2/14 Patients with T2D benefit from integrated multidisciplinary integrated care[9]. However, in Oujda, T2D management in public PHCCs is currently limited to general practitioners (GPs) and nurses, and treatment is predominantly focused on medication. Education about healthy food is done by GPs if they have time to do so at all. Exploring the views of HCPs is essential in order to help understand their preferences and suggestions for the improvement of T2D care. There is no published qualitative study that has examined the views of the HCPs about this phenomenon in Morocco. Therefore, this study aims to explore HCPs' perception of barriers, facilitators, attitudes, and benefits towards an integrated care approach of T2D management in Morocco. Procedure This study was reviewed and approved by the Comité d'Ethique pour la Recherche Biomédicale d'Oujda (CERBO) and participants signed informed consent before participating. An interview guide was developed and used to maintain the consistency of methods across groups (Additional file 1). The interviews were done in the French language but some participants combined this with Arabic dialect in the course of the interview. Focus group discussions lasted approximately 30 to 120 minutes and were moderated by a researcher with experience in qualitative methods. The focus group interviews were audiotaped and transcribed verbatim into the French language. Table 1: Distribution of HCPs in PHCCs for focus group Page 3/14 Page 3/14 Primary Healthcare Centers Doctors (n=5) Nurses (n=25) Oued Nachef 0 4 Nssar1 0 3 Sidi yahya 0 4 Al Andalouss 1 1 Mbasso 1 1 Boudir 1 5 Moulay driss 0 4 Lazaret 2 3 Primary Healthcare Centers Doctors (n=5) Nurses (n=25) Both doctors and nurses had working experience in T2D management for 6 or more months in a primary healthcare center. Besides the clinical task, some of the HCPs have administrative responsibility. Eligibility criteria included having experience of managing patients with T2D and practicing in the health facility where the interview is conducted. Both doctors and nurses had working experience in T2D management for 6 or more months in a primary healthcare center. Besides the clinical task, some of the HCPs have administrative responsibility. Eligibility criteria included having experience of managing patients with T2D and practicing in the health facility where the interview is conducted. Eligibility criteria included having experience of managing patients with T2D and practicing in the health facility where the interview is conducted. If a patient is hypo or hyperglycemic, there is no referral to a specialist (N). If a patient is hypo or hyperglycemic, there is no referral to a specialist (N). Data analysis Initially, all transcripts were written in the French language and before analysis, they were translated into English language using standardized procedures [11]. Using thematic analysis the textual data was analyzed in three steps. These include; (I) reading and re-reading transcripts until we became familiar with the views and language of participants; (II) Combining inductive and deductive approach to create a thematic framework (III) coding the data line-by-line against the themes and emerging sub-themes; and (IV) comparison and relational analysis of themes within cases of HCPs and PHCCs. Via face-to-face meetings, researchers identified the themes, which were discussed until consensus was reached. Transcripts in Microsoft Word format were imported into a qualitative research software package (NVIVO 12 Pro) to facilitate data management and analysis. Transcripts in Microsoft Word format were imported into a qualitative research software package (NVIVO 12 Pro) to facilitate data management and analysis. Page 4/14 Page 4/14 Results Health professionals identified barriers, facilitators, attitudes, and benefits of T2D treatment in primary health centers, which were grouped into three main categories: healthcare system, HCPs, and patients (Figure 1). We give longer appointments (D). Nurses have mentioned that one nurse is responsible for multiple tasks, which is time demanding.In addition, participants attribute shortage of staff to lack of recruitment and demotivated staff who are overdue for retirement but still in public service. Besides the lack of general practitioners in PHCCs, the doctors said there are no medical specialists to attend to patients with T2D requiring advanced care, and added that the nurses are not certified, diabetes specialists. Diabetic patients and health workers suffer due to too much workload (N). Diabetic patients and health workers suffer due to too much workload (N). Lack of enough staff to listen well (N). Lack of enough staff to listen well (N). We give longer appointments (D). Excessive workload and lack of human resources The disproportionate clinicians-to-patient ratio was mentioned by the participants as a major problem affecting proper individual medical consultations. They added that only a few doctors and nurses run the diabetes clinic and have to attend to long queues of patients beyond official working hours. Thus, often patients are given longer appointments for medical follow-up. Too many patients with diabetes are attending consultations, which does not allow adequate centered care for each patient (D). The nurses do not do their job due to lack of time, workload (N). Staffs are already old, sick, and demotivated (3 years above retirement age) almost 41 years of civil service (N). Specialists are no longer available in diabetic centers (D). Lack of adequate premises and diabetes education materials (N). The government cannot provide complete medical coverage for all the people. Lack of blood glucose meters. Routine assessment of lipid profile and HbA1c every three months are not done, because it's expensive. Lack of drugs. Patients do not buy drugs, because they are poor. Blood tests are not done and patients abandon their follow-ups (D). Restricted referral policy to diabetes centers The referral system is vague and patients find it difficult to make the transition from PHCCs to specialized healthcare centers. Participants noted that the Centre de Référence Préfectoral Diabétologie et Maladies Chroniques restrict referral of T2D patients to limited figures as opposed to the large number of patients with complications who need specialized care. Nurses mentioned that doctors exercise power autonomy by monopolizing the decision for referring patients to specialized care. More so, nurses perceived that Page 5/14 they always had to fill in too much patient information on the computer during consultations. They said it demands too much time and effort, as they have too many patients to attend to. they always had to fill in too much patient information on the computer during consultations. They said it demands too much time and effort, as they have too many patients to attend to. Centre de Référence Préfectoral Diabétologie et Maladies Chroniquesgives us a specific number of patients for referral (N). Lack of motivation in health professionals (D). Lack of adequate premises and diabetes education materials (N). Poor reimbursement policies and working environment At public hospitals, all HCP’s salaries are paid by the Ministry of finance. Doctors perceived their salary as low and not meeting their expectations. Participants described their working environment as having deteriorating working conditions. In addition, they mentioned that the universal medical coverage is inadequate due to lack of medical equipment and often the disrupted supply of free drugs. In addition, necessary medical investigations are left undone due to the poor economic status of the patients. Lack of motivation in health professionals (D). Ambiguous referral system and difficult for the patient to identify (D). It is only the doctors who recommend a referral if a patient is unstable or has complications (N) The Gap in doctor’s knowledge General practitioners expressed their incapability to manage elderly patients and complications. Questions about the general knowledge of T2D revealed that doctors perceived T2D as a chronic disease frequently found in young adults and the elderly population, and further highlighted heredity, obesity, and sedentary lifestyle as associated risk factors. They mentioned that treatment is non-insulin-dependent and lifestyle-oriented. Doctors only control the blood sugar and prescribe medication. When there are complications nothing can be done. They always come with complications (diabetic feet, amputation, or dialysis) (D). We do not target stable glycemic control in the elderly because it is difficult (D). We do not target stable glycemic control in the elderly because it is difficult (D). Page 6/14 Chronic disease, disease of the century. In all age categories above 30 years. Associated with heredity, obesity, or sedentary lifestyle. Clinically, a serious pathology. Requires assessment and multi-discipline monitoring (D). FACILITATORS AND BENEFITS ASSOCIATED WITH HEALTH SYSTEM MAN Participants suggested that an institutionalized referral system to a specialist, a simplified electronic health record, a continuous supply of free drugs, a diagnostic tools referral system, and recruitment of more HCPs into the civil service will augment structured T2D care in primary centers. Availability of drugs and equipment in adequate quantity and quality. Free routine medical investigations, for example, HbA1c and blood glucose. Recruit young competent staff. Simplify the health record database. Provide nurses just responsible for type 2 diabetes. Reduce appointment times (D). You need a nutritionist, dietetics, or nurse at CRD (in case of obesity) …. Ensure endocrinology consultation 1 time in 3 months in the primary medical centers (N). They added that there is a need to improve the working atmosphere. Favorable working conditions, air conditioning (N). BARRIERS ASSOCIATED WITH PATIENTS The participants expressed that beliefs about T2D among Moroccan patients are constructed out of cultural values and spiritual beliefs. Several patients believed that any illness including T2D comes from Allah (God) and he decides their fate with or without having T2D. This belief made them less likely to follow lifestyle changes advice. However, they do pay attention to getting free medicines. Patients do not accept their illness and do not adhere to diet advice, everything. Always the diabetic patients perceive higher HbA1c as caused by stress. They just need the drugs (D). Participants mentioned that patients’ willingness to adhere to treatment advice is negatively influenced by their poor socioeconomic status. Doctors mentioned the role of gender in terms of adherence to advice in favor of men. Nurses added that patients of advanced age did not engage in physical activity. Nurses said T2D patients were often prescribed antidepressant drugs, it influences their behaviors. Patients’ adherence to advice depends on socioeconomic status, culture, and education level (D). Men adhere to advice more than women (D). Men adhere to advice more than women (D). Aged have a problem especially with physical activity (N). Aged have a problem especially with physical activity (N). Diabetes patients always take psychotropic drugs (N). Continuous professional development program for staff that will provide diabetes education (D). Free medical consultation for these patients. Otherwise, patients will neglect the disease if not provided with their needs, everything (D). Therapeutic education should be done in the media and social networks for detailed diabetes education (D). Therapeutic education should be done in the media and social networks for detailed diabetes education (D). Locally adapted poster and teaching material (N). Locally adapted poster and teaching material (N). FACILITATORS AND BENEFITS ASSOCIATED TO HCPs AND PATIENTS Page 7/14 Participants recognized the need to engage in lifelong learning. They mentioned attending training would benefit continuously upgrading their skills. On the other hand, the participants perceived the provision of Page 7/14 free medical services as an approach that will promote diabetes self-management and therapy adherence. In addition, they stressed the need to use social media to create awareness and to promote diabetes education. free medical services as an approach that will promote diabetes self-management and therapy adherence. In addition, they stressed the need to use social media to create awareness and to promote diabetes education. Continuous professional development program for staff that will provide diabetes education (D). Continuous professional development program for staff that will provide diabetes education (D). Discussion This study provided an in-depth understanding of the barriers and facilitators for a integrated approach to T2D management. The barriers were excessive workload, insufficient medical consultation time, and lack of human resources. The views of nurses, doctors, and health service managers differ, so determining the optimal doctor-to-patient ratio is a complex matter [12]. For too many GPs, the workload has become unmanageable, so patients don't receive adequate care. Moreover, the pressure leads to burn-out and the intention to leave the profession earlier than planned. This will certainly have a major impact on the time patients have to wait for a follow-up medical appointment [13]. Although there is insufficient evidence to either support or resist a policy of increasing consultation lengths, longer consultations may facilitate integrated care and offer sufficient time to explain treatment options to patients [14]. More so, nurses described their work as multitasking demanding a lot of time. Indeed, nurses’ cognitive load is exceptionally heavy and the environment surrounding them is fast-paced and unpredictable.Nurses can manage interruptions and multitask well, but the potential risk for errors increases with the workload, hence strategies to decrease interruptions are needed [15]. Doctors raised concerns about the lack of easily available specialists to attend to patients suffering from complications. Also, they described the referral system as unclear and difficult for patients to make a transition from PHCCs to specialized care. Moreover, only a limited number of patients with T2D are accepted in diabetes referral centers. Literature on the referral practices of GPs for diabetes shows that factors influencing the referral threshold include multiple complications, the presence of comorbidities, uncertainty concerning medical management, and the need for self-management education [16, 17]. The inability to gain timely access to specialized diabetes consultation teams may contribute to GP’s and patients’ frustration and sub-optimal disease management [10]. In this specific study, nurses perceived that it is the sole responsibility of doctors to refer patients to specialized care. However, models of nurse-led T2D clinics exist worldwide. Hence, this needs to be addressed in order to improve the quality of diabetes care. Evidence shows that diabetes nurse Page 8/14 Page 8/14 specialists have a key role in supporting and coordinating the integrated care management of T2D through nurse-led clinics in primary care. They facilitate the delivery of integrated care within the PHCCs and the cooperation between health care settings and specialists [18, 19]. Discussion An integrated approach for T2D management is effective in significantly reducing glycated hemoglobin levels, increasing treatment compliance and it is cost-effective [20]. Including specialist diabetes nurses, dieticians, physiotherapists and psychologists would allow GPs and nurses to focus on their core tasks. GPs and Nurses found it difficult to include physical activity support and healthy lifestyle advice in aT2D care program. But integrating physiotherapists into the interdisciplinary leads to the opportunity to include specialized diabetes physical activity care to elderly patients [21]. In our study, we were not able to explore how GPs and nurses advise patients to engage in physical activity since triangulation between the quotes of nurses and GPs was not done.. The introduction of an electronic health record system (EHR) is an important development to facilitate the processing of large amounts of patient data. However, in our study nurses perceived EHR as an additiona workload in T2D care because they had to input numerous data during medical consultations. This finding is quite essential given the current trend of using computer applications in diabetes management, and thus this contributes to the strength of this study. A previous study [22] that examines the emerging practices of the use of EHR in diabetes care reported that there is a mismatch between EHR designs and diabetes care practices. Some health professionals perceived EHR as a source of increase in caregivers' administrative time, while others described EHR as a tool that modestly decreases the clinical documentation time. Therefore, it is recommended that system designers first make overviews of the necessary information about diabetes patients in order to develop systems that can be used optimally during clinical consultations. Doctors that participated in the focus group discussions indicated that they were not satisfied with their salaries. Clinical management, according to recommended guidelines, at the level of primary care improves outcomes for patients with diabetes but it is linked to incentives for health professionals. In the United Kingdom, evidence has shown that incentive models have spurred some improvements in process outcomes and achievement of cholesterol, blood pressure, and HbA1C targets [23]. To improve subsidized diabetes care, Morocco has included the prevention and control of diabetes as a priority program in its health action plan 2012-2016. Still, diabetes care is limited. In 2011, the number of diabetic patients supported by the Moroccan Ministry of health was 460,000 representing only 33% of the known diabetics [24]. Strengths and limitations To the best of our knowledge, this study was the first in Morocco to assess perceived barriers, benefits, facilitators, and attitudes of health professionals towards T2D management. Nevertheless, our study has some limitations. A convenience sample was used to select the participants possibly leading to some missed respondents. The data was translated into English affecting the trustworthiness of the original data due to an interpretation of the translator. Also, there was no member checking, and we did not include perceptions of patients. However, the specific findings in this study provide essential information to strengthen T2D care in this region. We also reached data sufficiency after eight focus groups, giving sufficient richness and depth of the data. To the best of our knowledge, this study was the first in Moroc facilitators, and attitudes of health professionals towards T2D some limitations. A convenience sample was used to select th missed respondents. The data was translated into English aff data due to an interpretation of the translator. Also, there was include perceptions of patients. However, the specific findings to strengthen T2D care in this region. We also reached data su sufficient richness and depth of the data. Discussion Patients who have no health care insurance receive high medical bills. Those who cannot afford or are financially unable do not use the health services or do not adhere to the treatment. [25]. Islam is predominantly the religion practiced in Morocco and some Muslims with T2D believe that health and illness are part of Allah's plan determining their fate and time of death with or without having T2D. Consequently, patients are less likely to clearly understand the disease and the urgency for lifestyle changes. Alsairafi et al.,[26] reported that many patients believed that only God can cure diabetes and not doctors or medicines. However, it is well known that severe untreated cases of diabetes can lead to death, Page 9/14 Page 9/14 and there are different verses of the Holy Quran (the Islamic divine book) and Prophet’s sayings that stress disease prevention and treatment. Participants in our study perceived that elderly patients do not engage insufficient physical activity. However, there is functional heterogeneity because some older patients are highly functional, some are disabled and frail, and many are between these two extremes [27]. Hence, tailored programs can address this specific population. CONCLUSIONS AND IMPLICATIONS Our findings indicate a need for an adapted patient-centered multidisciplinary team and an integrated care approach. The importance of political will and the possibility to ensure adequate universal health coverage, and well-equipped PHCCs with synchronized secondary care service should be emphasized. In addition, including dieticians, physiotherapists and occupational therapists with expertise in diabetes are crucial for establishing lifestyle-changing interdisciplinary programs. A discussion of our findings with the health policy authorities of the Ministry of Health of Morocco could lead to suggestions to strengthen the organization of T2D care to meet the needs of patients. There is a need for changes in the behavior of professionals towards less authoritarian styles of medical decision-making and more patient-centered care. The characteristics of the patient-care provider relationship that affect reluctance to change lifestyle also deserve further research. Ethics approval and consent to participate This study was ethically approved and reviewed by the Comité d'Ethique pour la Recherche Biomédicale d'Oujda (CERBO). Consent for publication Participants signed informed consent before participating in the study. Page 10/14 Authors' information (optional) ”Not applicable” Funding The City of Antwerp: Research mobility grant for the first author. The City of Antwerp: Research mobility grant for the first author. The Government of Flanders, Mastermind Scholarship (698261): Research grant for the first author. VLIR-UOS South Initiative project funding (SI2019- S EL053): Study and collection, analysis, and interpretation of data and writing manuscript. Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Authors' contributions DV, BVR, SBEM, AM, and AZ contributed to the study conception and design. DV, AM, and USD performed data acquisition, analysis, interpretation of data, and drafting the article. DV, PVR and AM revised the article critically for important intellectual content and final approval of the version to be published. All authors read and approved the final manuscript. Acknowledgements ”Not applicable” Authors' information (optional) Competing interests The authors declare that they have no competing interests/ References 1. Jaacks LM, Siegel KR, Gujral UP, Narayan KMV. Type 2 diabetes: A 21st century epidemic. Best Practice & Research Clinical Endocrinology & Metabolism. 2016;30(3):331-43. doi:https://doi.org/10.1016/j.beem.2016.05.003. 1. Jaacks LM, Siegel KR, Gujral UP, Narayan KMV. Type 2 diabetes: A 21st century epidemic. Best Practice & Research Clinical Endocrinology & Metabolism. 2016;30(3):331-43. doi:https://doi.org/10.1016/j.beem.2016.05.003. 2. Diabetes IFo. 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Canadian Journal of Diabetes. 2015;39(1):83-7. doi:10.1016/j.jcjd.2014.06.002. 23. Latham LP, Marshall EG. Performance-Based Financial Incentives for Diabetes Care: An Effective Strategy? Canadian Journal of Diabetes. 2015;39(1):83-7. doi:10.1016/j.jcjd.2014.06.002. 24. Dinar Y, Belahsen R. Diabetes Mellitus in Morocco: Situation and Challenges of Diabetes Care. Journal of Scientific Research and Reports. 2014;3:2477-85. doi:10.9734/jsrr/2014/10287. 25. Yasmin F, Ali L, Banu B, Rasul FB, Sauerborn R, Souares A. Understanding patients’ experience living with diabetes type 2 and effective disease management: a qualitative study following a mobile health intervention in Bangladesh. BMC Health Services Research. References 2020;20(1):29. doi:10.1186/s12913-019-4811-9. 26. Alsairafi ZK, Taylor KM, Smith FJ, Alattar AT. Patients' management of type 2 diabetes in Middle Eastern countries: review of studies. (1177-889X (Print)). 27. Blaum CS. Management of diabetes mellitus in older adults: are national guidelines appropriate? Journal of the American Geriatrics Society. 2002;50(3):581-3. doi:10.1046/j.1532- 5415.2002.50130.x. 27. Blaum CS. Management of diabetes mellitus in older adults: are national guidelines appropriate? Journal of the American Geriatrics Society. 2002;50(3):581-3. doi:10.1046/j.1532- 5415.2002.50130.x. Figures Figure 1 Conceptual framework of type 2 diabetes treatment barriers, facilitators, attitudes and benefits derived from general practitioners and nurses in Oujda Conceptual framework of type 2 diabetes treatment barriers, facilitators, attitudes and benefits derived from general practitioners and nurses in Oujda Figures Page 13/14 Page 13/14 gure 1 Figure 1 Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. ADDITIONALFILE1.docx ADDITIONALFILE1.docx ADDITIONALFILE1.docx Page 14/14
https://openalex.org/W1632887891	http://cds.cern.ch/record/849078/files/daq-conf-2005-028.pdf	English	null	Editor for Remote Database used in ATLAS Trigger/DAQ	AIP conference proceedings	2,006	cc-by	1,703	Editor for Remote Database used in ATLAS Trigger/DAQ Abstract. This article describes the RDB Editor, the application for user-friendly viewing and edit- ting of the contents of the RDB database used in the ATLAS Trigger/Data Acquisition. The appli- cation provides convenient navigation between huge number of stored objects and the possibility of developing plug-ins that ﬁt the special needs of different working groups. Keywords: ATLAS, HLT, Remote Database PACS: 07.05.Hd REMOTE DATABASE Many of the components of the ATLAS T/DAQ system use a common database for storing their conﬁguration data. Recently, the database used is the Remote Database[1] (RDB). This database uses XML ﬁles for schema deﬁnition and data storage, and an RDB server for accessing them. The schema ﬁles describe the class structure of the database. It states what is the relation between particular classes (they can be inherited, for example), limits the types and values for particular attributes, lists the possible links to other classes, etc. The data themselves are stored in the database in form of objects, each of them implementing particular class. The objects already have concrete values assigned to attributes and relationships. There is a pair of tools for direct access to the XML ﬁles and altering the database schema or the data themselves (OKS Schema Editor and OKS Data Editor, see Chapter 3 in [1]). The application described in this article uses the rdb_server program and accesses the XML ﬁles via this one additional layer. RDB EDITOR Because a huge amount of objects is foreseen in the RDB in the future and each project using the database will need to conveniently navigate between them, Java application called “RDB Editor” was developed. The RDB Editor provides the user with an easy-to- use GUI and implements the following features: • easy navigation between already visited objects. • several views on history of visited objects. There is also the possibility of searching in the history and of sorting it according to the name of the object or time. • bookmarks, including managing. • write support. • write support. • graphical viewer for special classes of objects. As an example, the viewer of EFD_Configuration class is provided. • graphical viewer for special classes of objects. As an example, the viewer of EFD_Configuration class is provided. The application is written in Java to be possibly able to take advantage of integra- tion into the ATLAS Online Software as a separate panel. The Eclipse Development Platform1 was used during the development for analyzing, coding, and debugging. 1 http://www.eclipse.org Internal Structure The structure of the RDB Editor application is outlined in Figure 1. The lowermost rdbComm layer cooperates with the RDB interface provided by the rdb_server, the ConfCore component implements the higher-level functionality including bookmarks and histories support and provides user-friendly interface for RDB access. Finally, the topmost layers are clients, both general and specialized. FIGURE 1. Software layers in the RDB Editor. FIGURE 1. Software layers in the RDB Editor. The rdbComm layer is responsible for the lowest level communication with the database. It provides methods for accessing the RDB for both reading and writing. There is also a possibility to read the schema and data separately. There are also several helper classes deﬁned in this layer. Their full description, including description of their members and methods, can be found in the Javadoc documentation[2]. Among the most important ones are Attribute, which encapsu- lates the properties of particular attribute, and AttributeValue, which represents name and value of a particular attribute. The confCore layer just adds some functionality to the underlaying layer, otherwise it just forwards the database access requests to the rdbComm. This added functionality is, for example, support for bookmarks, history of visited objects, and navigation track. There is the GUI appearance and functionality deﬁned in the rdbMng. The main class here contains the main method and logic responsible for program running, the rest of classes deﬁned in this layer are just helper GUI classes. For example, there are classes that provide the additional functionality of the ComboJTable, like the hypertext nature of object identiﬁers in the table (in relation- ship lines) or the ability of editing the values. The GUI deﬁned by this set of classes is able to browse through RDB database con- taining any set of objects. The easy-to-use navigation through the database is supported for all abjects deﬁning the relationships and the histories are available all the time. It is universal RDB viewer and writer. The application also supports addition of custom graphical viewers for displaying speciﬁc object relationships which are not easily understandable with the default viewer. As an example, the viewer for EFD_Configuration instances was implemented. The objects of this class represent the conﬁguration of Event Filter Dataﬂow, the way the event data are routered between the SFIs and SFOs. Internal Structure Many different tasks may deal with the event in-between, they may for example log some data, make some computation, move the data to the trash, etc. Browsing of the EFD_Configuration in the textual view can be quite compli- cated, as loops in the event dataﬂow may occur or the net of the processing tasks may be very large. Therefore, the graphical plug-in may help with understanding of the dataﬂow net. When the user displays an object of the EFD_Configuration class, the second panel in the attribute-area appears, showing the graphical representation of the object showing the user the conﬁguration information he needs in a comprehensive way. The conﬁguration graph is implemented using the JGraph[3] component. This is a framework for creating Java applications working with various graphs or diagrams. Graphical User Interface As one can see in Figures 2 and 3, the application window can be divided into three main sections. There is a detachable toolbar on the top, tabbed histories display on the left, and potentially tabbed properties viewer which occupies the rest of the window. There are also two separate windows designed to provide the GUI for bookmarks managing and preferences setting. The menu in the main window of the application provides access to preference settings, database connection command, and Bookmarks options. In the preferences, the user can store the value used for ‘Home’ object and the database name used for connecting to the rdb_server (other possibility is the command-line parameter). The bookmarks submenu offers organizing and saving/loading of the bookmarks. The detachable toolbar under the menu contains navigation buttons, object selection items, and ‘Show’ and ‘Write’ buttons. The object selection comboboxes allow the user to select the class and consequently an object of that class. The ‘Show’ button actually loads the selected object into the properties panels and the ‘Write’ button writes the changes made to the attributes or relationships to the database. FIGURE 2. Textual view on the EFD_Configuration object. FIGURE 3. Graphical view on EFD_Configuration object. There are several views on the history of visited objects in the program. The defa ne, ‘History’, logs the name of the visited object and the time it was visited. This pa lows the user to see what object was displayed at any point in the history. The ‘Last Visited’ tab works as sort of live bookmarks. One can see the list of st visited objects there with no duplicate records and no timestamps. There is als ibilit f hi d ti th hi t i FIGURE 2 Textual view on the EFD Configuration object FIGURE 2. Textual view on the EFD_Configuration object. FIGURE 2. Textual view on the EFD_Configuration object. FIGURE 3. Graphical view on EFD_Configuration object. FIGURE 3. Graphical view on EFD_Configuration object. There are several views on the history of visited objects in the program. The default one, ‘History’, logs the name of the visited object and the time it was visited. This panel allows the user to see what object was displayed at any point in the history. There are several views on the history of visited objects in the program. SUMMARY The application described here is meant to help people using the RDB database for storage of huge numbers of object to stay oriented. It offers comprehensive way of navigation and the possibility of developing specialized viewers. Scalability The component EFDConfGraph is self-contained part of the program. Similar func- tional blocks can be developed by members of any other group which is storing its con- ﬁguration data in the rdb database. The interconnection of the newly developed specialized part and the core program involves some minor changes of the program source code and a recompilation. The developer of such a plug-in (graphical or not) may and should use the infrastructure provided by lower levels of the RDB Editor application; namely the standardized access to the database via the confCore interface is a must to keep the histories and track of visited objects up to date. Graphical User Interface The default one, ‘History’, logs the name of the visited object and the time it was visited. This panel allows the user to see what object was displayed at any point in the history. The ‘Last Visited’ tab works as sort of live bookmarks. One can see the list of 15 last visited objects there with no duplicate records and no timestamps. There is also a possibility of searching and sorting the histories. The largest part of the application window is occupied by the properties view. Figure 2 shows the textual view on the properties of the EFD_Configuration object. The relationships values act as hypertext links. The graphical plug-in for displaying the objects of the EFD_configuration class is shown in Figure 3. The displayed tasks are also hypertext links; hovering over an object shows detailed information in a tooltip. ACKNOWLEDGMENTS The work was done during Marie-Curie fellowship of Jan Valenta at CPPM Marseille. Thanks go also to ATLAS T/DAQ collaboration for developing the whole Trigger and Data Acquisition system and maintaining great documentation. 3 http://cern.ch/jvalenta/rdbMng 4 4 http://www.jgraph.com 2 http://atddoc.cern.ch/Atlas/DaqSoft/components/configdb/docs/ confdb-ug/2.3/pdf/ConfDB.pdf 3 2 http://atddoc.cern.ch/Atlas/DaqSoft/components/configdb/docs/ confdb-ug/2.3/pdf/ConfDB.pdf 3 http://cern.ch/jvalenta/rdbMng 4 http://www.jgraph.com REFERENCES 1. I. Soloviev: Conﬁguration Databases User’s Guide, ATLAS DAQ Technical Note 1352. 2. C. Meessen, F. Touchard, J. Valenta: Java application for rdb management, Javadoc documentatio 4 2. C. Meessen, F. Touchard, J. Valenta: Java application for rdb management, Javadoc doc 4 3. JGraph, Ltd.: The Java Graph Visualization Library4.
https://openalex.org/W2980016529	https://www.frontiersin.org/articles/10.3389/fbioe.2019.00250/pdf	English	null	Identification and Analysis of Long Repeats of Proteins at the Domain Level	Frontiers in bioengineering and biotechnology	2,019	cc-by	8,971	Edited by: Shandar Ahmad, Jawaharlal Nehru University, India Reviewed by: Michael Gromiha, Indian Institute of Technology Madras, India Vladimir N. Uversky, University of South Florida, United States Tadashi Satoh, Graduate School of Pharmaceutical Sciences, Nagoya City University, Japan Bratati Kahali, Indian Institute of Science, India Correspondence: Samuel Selvaraj selvarajsamuel@gmail.com Keywords: long repeats, protein, domain, protein family, enzyme and non-enzyme classes, structural fold Specialty section: This article was submitted to Bioinformatics and Computational Biology, a section of the journal Frontiers in Bioengineering and Biotechnology ORIGINAL RESEARCH published: 08 October 2019 doi: 10.3389/fbioe.2019.00250 Identiﬁcation and Analysis of Long Repeats of Proteins at the Domain Level David Mary Rajathei, Subbiah Parthasarathy and Samuel Selvaraj Department of Bioinformatics, School of Life Sciences, Bharathidasan University, Tiruchirappalli, India David Mary Rajathei, Subbiah Parthasarathy and Samuel Selvaraj* Department of Bioinformatics, School of Life Sciences, Bharathidasan University, Tiruchirappalli, India Amino acid repeats play an important role in the structure and function of proteins. Analysis of long repeats in protein sequences enables one to understand their abundance, structure and function in the protein universe. In the present study, amino acid repeats of length >50 (long repeats) were identiﬁed in a non-redundant set of UniProt sequences using the RADAR program. The underlying structures and functions of these long repeats were carried out using the Gene3D for structural domains, Pfam for functional domains and enzyme and non-enzyme functional classiﬁcation for catalytic and binding of the proteins. From a structural perspective, these long repeats seem to predominantly occur in certain architectures such as sandwich, bundle, barrel, and roll and within these architectures abundant in the superfolds. The lengths of the repeats within each fold are not uniform exhibiting different structures for different functions. We also observed that long repeats are in the domain regions of the family and are involved in the function of the proteins. After grouping based on enzyme and non-enzyme classes, we observed the abundant occurrence of long repeats in speciﬁc catalytic and binding of the proteins. In this study, we have analyzed the occurrence of long repeats in the protein sequence universe apart from well-characterized short tandem repeats in sequences and their structures and functions of the proteins at the domain level. The present study suggests that long repeats may play an important role in the structure and function of domains of the proteins. Keywords: long repeats, protein, domain, protein family, enzyme and non-enzyme classes, structural fold INTRODUCTION Amino acid repeats are ubiquitous in protein sequences that often correspond to structural and functional units of proteins. The length of these repeats varies considerably from shorter units of homo repeats of single amino acid (Jorda and Kajava, 2010), oligopeptide repeats of 2–20 residues (Fraser and MacRae, 1973) and solenoid repeats of 20–40 residues to larger repetitions of length >50 called domain repeats (Andrade et al., 2001). These repeats occur as a single pair or as multiple copies in a tandem/non-tandem manner that are useful for structural packing or for one or more interactions with ligand (Katti et al., 2000; Luo and Nijveen, 2014). It has been observed that many proteins of length >500 contain internal repeats, suggesting the importance of repeats in producing larger proteins (Marcotte et al., 1998). However, these repeats possess weak identities due to extensive divergence, but retain similar folds and functions of the proteins (Holm and Sander, 1993). It has also been found out that long stretches of perfect repetitions are infrequent in protein Received: 18 April 2019 Accepted: 16 September 2019 Published: 08 October 2019 Data Collection p Analysis of larger proteins has demonstrated that signiﬁcant portions of proteins are composed of domains. They are the conserved parts of proteins which can fold and function independently. The folded domains can either serve as modules for building up large assemblies or provide speciﬁc catalytic enzyme functions or bindings of the proteins. It has been found that repeats of a length >50 residues often correspond to conserved regions that are present in proteins as single or multiple copies for the function of the proteins (Hemalatha et al., 2007). Our analysis of sequence repeats of the proteins with known 3D structures in the PDB (Berman et al., 2014) has shown that they retain similar folds in spite of divergences, in order to conserve the structure and function of the proteins and, repeats that are in the single/two domains from the same family contain conserved motifs for the function of the proteins (Mary Rajathei and Selvaraj, 2013). Further, the conservation of inter- residues interactions in domain repeats have been analyzed in terms of long-range contact, surrounding hydrophobicity and pair-wise interaction energy (Mary et al., 2015). A database IR- PDB for repeats in the sequence of the proteins in the PDB has been developed for the analysis of impact of repeats in proteins (Selvaraj and Rajathei, 2017). A collection of 555,100 proteins along with their assigned UniProt ID, amino acid sequence, protein name, protein family, enzyme function, and non-enzyme functions such as DNA binding, calcium binding, metal binding, and NP binding, as well as other annotation of the sequences from the databases of Pfam, Gene3D, PDB, and DisProt, was downloaded from UniProtKB/Swiss-Prot (UniProt Consortium T, 2017) and stored in a ﬁle. The Pfam is a database of protein domain families that assigns the domains, as well as their functional regions (Finn et al., 2014). Gene3D (Lewis et al., 2018), is a database that assigns the structure of the protein according to CATH hierarchy of class, architecture and fold in numerical values (Dawson et al., 2017). At the class level (C), the numerical value 1 is for all alpha class, 2 for all beta and 3 for a mixture of alpha and beta. Likewise, the numerical values are assigned for Architecture level (A) based on secondary structure arrangement in 3-D space and for Topology/Fold level (T) based on the connection of secondary structural elements. Citation: Rajathei DM, Parthasarathy S and Selvaraj S (2019) Identiﬁcation and Analysis of Long Repeats of Proteins at the Domain Level. Front. Bioeng. Biotechnol. 7:250. doi: 10.3389/fbioe.2019.00250 October 2019 \| Volume 7 \| Article 250 Frontiers in Bioengineering and Biotechnology \| www.frontiersin.org Long Repeats in Protein Sequences Rajathei et al. armadillo/heat etc. However, there has been no survey of repeats of length>50 in the UniProt sequences, which may provide insights into their role in the structure, function and evolution of the proteins. In the present study, we have analyzed the occurrence of long repeats and their underlying structures and functions in a non-redundant set of UniProt sequences. Since repeats of size exceeding 50 residues are large enough to fold independently into stable domains (Kajava, 2012), we used Gene3D for structural domains, Pfam for functional domains and enzyme and non-enzyme functions for speciﬁc catalytic and binding for their structure and function of long repeats proteins. It was found that long repeats occur in about 23% of the considered proteins. Analysis of the structure of long repeats reveals that these repeats are predominantly observed in the structural folds of sandwich, bundle, barrel and roll. We observed that repeats in the domains for the function of the proteins. Further, we observed that long repeats tend to occur both in enzyme and non-enzyme functions of proteins. While long repeats are found in all the major enzyme classes, these are more abundant among both ligases and isomerases. Among the non-enzyme proteins, such as DNA binding, metal binding, calcium binding, and Nucleotide binding (NP), these repeats are observed more in Nucleotide binding and DNA binding proteins. The present analysis shows that the occurrence of long repeats and their structures and functions of the proteins at the domain level. sequences even though they are folded into recurrent structural motifs (Turjanski et al., 2016). Many methods and algorithms, such as Fourier transformation, short string extension, sequence- sequence alignment, and sequence proﬁles comparison have been introduced for the identiﬁcation of such diverged sequence repeats with insertion and deletion without prior knowledge. Citation: Web based servers such as the Internal Repeat Finder, RADAR, REPRO, TRUST, XSTREAM, HHRepID, T-REKS, and PTRStalker (Pellegrini et al., 1999, 2012; George and Heringa, 2000; Heger and Holm, 2000; Szklarczyk and Heringa, 2004; Newman and Cooper, 2007; Biegert and Söding, 2008; Jorda and Kajava, 2009) have been developed by implementing the above techniques to detect amino acid repeats in proteins. Earlier, proteins containing homo repeats (Jorda and Kajava, 2010), ﬁbrous repeats (Fraser and MacRae, 1973) and diﬀerent well-characterized repeats types, namely tetratricopeptide, leucine-rich, ankyrin and armadillo/heat etc. (Fraser and MacRae, 1973; Yoder et al., 1993; Groves and Barford, 1999; Kobe and Kajava, 2001), possessing diﬀerent structures and functions have been analyzed (Andrade et al., 2001). Further, short units of repeats in tandem that form repeats in the structural folds of solenoids (α, β, α/β), β-trefoil (Murzin et al., 1992; Ponting and Russell, 2000), β-prisms (Chothia and Murzin, 1993; Bourne et al., 1999), and β-propellers (Bork and Doolittle, 1994; Neer et al., 1994) have been reviewed (Kajava, 2012). Recently, a detailed analysis and classiﬁcation of β-hairpin repeat structures has been carried out (Roche et al., 2017). Also, it has been pointed out that short tandem repeats accumulate in the intrinsically disordered regions (IDR) (van der Lee et al., 2014) and play an important role in protein interactions and stability (Tompa, 2012; Habchi et al., 2014). Finding Sequence Repeats of the Proteins Using RADAR proteins were extracted using a Perl program. Then, the name of class, architecture and fold of the protein was found out by using CATH search and grouped according to their name for the further analysis of architecture and fold of the protein with repeats. The presence of internal repeats in each protein sequences was identiﬁed using the repeat detection program RADAR (Heger and Holm, 2000), which was downloaded from the URL (https:// sourceforge.net/projects/repeatradar). The RADAR program is eﬃcient for ab initio detection of repeats of length >15 in a single sequence by aligning the sequence against itself, as well as by generating the sequence proﬁle using multiple sequence alignment. RADAR evaluates the statistical signiﬁcance of the observed repeats by measuring a Z-score for each repeat unit (McLachlan, 1983; Heringa and Argos, 1993). The Z-score of a repeat unit is the number of standard deviations of the repeat unit score above the mean. The score of each unit is determined from a proﬁle derived from the multiple alignment of repeat unit without considering end-gaps. Repeats with Z-scores threshold of > 6 are reported by the RADAR program. An in-house Perl program that incorporated the RADAR executable was written to detect internal repeats of all sequences in the dataset in a single run. Proteins containing repeats of length >50 were considered for further analysis. Finding the Functional Domains of Long Repeats Proteins p The UniProt ID’s of long repeat proteins were extracted and their assigned Pfam domains of the sequences were identiﬁed. The domain regions and their functional residues information of the proteins were found out using Pfam database search (Finn et al., 2014), and repeats in the domain regions were identiﬁed by manual search. The level of similarity of the repeats within a protein and within a protein family was found out in terms of % sequence identity through using the Needleman–Wunsch algorithm (Needleman and Wunsch, 1970) implemented in the ggsearch36 program of the FASTA-36.3.5b package (Henikoﬀ and Henikoﬀ, 1992). Needleman–Wunsch alignment scores were calculated using the BLOSUM50 scoring matrix (Pearson, 2000) with a penalty of −12 for gap opening and −2 for gap extension. Further, the repeats in domains of the proteins were also analyzed for their functional involvement at the structure of the proteins using the server PDBsum by giving PDB ID as input (Laskowski et al., 2018). Data Collection The PDB ID’s of the 3D structure known proteins were obtained from the PDB database (http://www. rcsb.org/pdb/home/home.do). The intrinsic disordered regions of the proteins that were extracted from the literature are available in the DisProt database (Piovesan et al., 2017). A non- redundant representative set of 126,945 sequences that share <50% sequence identity was obtained by clustering the 555,100 sequences using the web server CD-HIT (Fu et al., 2012). The overall work-ﬂow is summarized as a ﬂowchart (Figure 1). The widely used sequence database UniProtKB (UniProt Consortium T, 2017) contains more than 500,000 sequences that are annotated with well-characterized repeats of tetratricopeptide, leucine-rich repeats, ankyrin, and October 2019 \| Volume 7 \| Article 250 Frontiers in Bioengineering and Biotechnology \| www.frontiersin.org 2 Rajathei et al. Long Repeats in Protein Sequences FIGURE 1 \| Flow diagram of identiﬁcation and analysis of Long repeats from non-redundant set of UniProt sequences. m of identiﬁcation and analysis of Long repeats from non-redundant set of UniProt sequences. Abundance of Proteins Having Long Repeats The structural class, architecture and fold of the 14,176 proteins (48%) have been found out using structural domain based Gene3D assignments. Among these, some proteins are having two or more Gene3D assignments. In this study, 10,504 proteins that contained a single Gene3D assignment were considered for further analysis (Supplementary Data File 2). For example, Annexin A1 (P04083) protein contains a single Gene3D assignment of 1.10.220, which means that this protein belongs to class alpha (1) of orthogonal bundle architecture (10) with Annexin V domain fold (220). p The presence of amino acid repeats of length >15 was found out in 85,726 (67%) out of non-redundant set of 126,945 UniProt protein sequences (Supplementary Data File 1). The long repeats were found out in 29,768 (35%) proteins. These repeats are present as a single pair or multiple copies of repeats in tandem/non-tandem manner. For example, N- acetylmuramoyl-L-alanine amidase Rv3717 protein (UniProt ID: I6Y4D2) of length 241 contains a single pair of tandem repeats of length 96 in the continuous region of 12–116/118–226. Complement control protein C3 (P68639) of length 263 has three copies of repeats of length 55 in tandem (81–142/143– 200/201–254), whereas Transcriptional regulatory protein TyrR (UniProt ID: P44694) of length 318 contains a single pair of non-tandem repeats of length 76 in the discontinuous region of (19–99/200–279). The length of the repeats varies in the range of 51–1759 and lengths of >1,000 are mostly found out in enzyme proteins. For example, the non-ribosomal peptide synthetase 1 (Q4WT66) of sequence length 6,269 Finding the Enzyme and Non-enzyme Functions of Long Repeats Proteins contains repeats of length 1,546 (277–905/906–2,334/2,335– 3,465/3,466–4,590/4,593–5,634). Through the analysis of length distribution of long repeats, as well as their repeat number distribution of long repeats against the number of proteins (Figure 2), we observed that the lengths <200 are observed in more than 90% of the proteins with an average of 100 residues, and repeated in 2–5 number of times with repeat numbers of 2 (61%) and 3 (26%) in most of the long repeat proteins. The Z-score values of the repeats were extracted and found that 74,089 out of 74,154 repeat units have Z- scores >6. Among these, 66,400 repeat units have Z-scores of >20. This suggests that most of the observed repeats are statistically signiﬁcant. The assigned enzyme numbers (EC) of long repeats proteins were extracted. The EC number of the protein at the ﬁrst level corresponds to seven enzyme classes of Oxidoreductases (EC 1), Transferases (EC 2), Hydrolases (EC 3), Lyases (EC 4), Isomerases (EC 5), Ligases (EC 6), and ﬁnally, Translocases (EC 7). The enzyme numbers were extracted and grouped according their numbers for further analysis. The non-enzyme proteins that are assigned with DNA binding, calcium binding, metal binding and NP binding were also extracted and grouped according to their name. Finding the Structure of Long Repeats Proteins The UniProt ID’s of proteins having long repeats were extracted and their Gene3D structural domain-based assignments of the October 2019 \| Volume 7 \| Article 250 Frontiers in Bioengineering and Biotechnology \| www.frontiersin.org 3 Rajathei et al. Long Repeats in Protein Sequences FIGURE 2 \| The plotting of number of proteins against the distribution of long repeats of length >50 in the range of <100, 101–200, 201–300, 301–400, 401–500, and>500 shows that most of the longrepeat lengths fall in the range of <200 (A) and repeat number distribution of long repeats shows that repeat numbers of 2 and 3 in most of long repeat proteins (B). FIGURE 2 \| The plotting of number of proteins against the distribution of long repeats of length >50 in the range of <100, 101–200, 201–300, 301–400, 401–500, and>500 shows that most of the longrepeat lengths fall in the range of <200 (A) and repeat number distribution of long repeats shows that repeat numbers of 2 and 3 in most of long repeat proteins (B). FIGURE 2 \| The plotting of number of proteins against the distribution of long repeats of length >50 in the range of <100, 101–200, 201–300, 301–400, 401–500, and>500 shows that most of the longrepeat lengths fall in the range of <200 (A) and repeat number distribution of long repeats shows that repeat numbers of 2 and 3 in most of long repeat proteins (B). Finding the Enzyme and Non-enzyme Functions of Long Repeats Proteins Analysis of Long Repeats for Structural Repeats p The long repeats in proteins with known 3-D structure (as available from UniProt annotation) were analyzed for structural repeats. The proteins with tandem repeats were found out and analyzed at the structural level. We observed long tandem repeats form structural repeats in the folds of up-down and orthogonal bundle of α-class, Immunoglobulin, Jelly Roll and OB fold of β-class, Rossmann fold, TIM barrel, α/β plait, and UB roll of α/β class. Figure 5 shows the structural repeats of the proteins in the folds of up-down and orthogonal bundle of α-class, Immunoglobulin, Jelly Roll and OB fold of β-class, Rossmann fold, TIM barrel, α/β plait, and UB roll of α/β class. Further, we found out that the lengths of the repeats are not uniform and vary considerably within each fold. Figure 6 shows the considerable variation in lengths, as well as in the secondary structures of diﬀerent proteins possessing the Rossmann fold that usually contains βαβαβ secondary structure arrangements. The Desulfovibrio vulgaris CbiK(P) Cobaltochelatase (PDB ID: 2XVY) contains two repeats of βαβαβαβ secondary structure arrangement of length 103 (Figure 6A) (Malay et al., 2009), whereas, another protein Thermoplasma volcanium Phosphoribosyl pyrophosphate synthetase (PDBID: 3MBI) contains two repeats Analysis of Long Repeats at the Fold Level The existence of repeats in diﬀerent folds of sandwich, bundle, barrel and roll architectures was found out. Repeats were observed in 84 out of 287 folds in orthogonal bundle and 32 out of 101 folds in up-down bundle of α class. At the β class, 12 out of 43 folds in β sandwich, 18 out of 48 folds in β barrel, and 13 out of 40 folds in β roll architecture of the proteins were having repeats. Under α/β class, repeats in 47 out of 126 folds under 3-layer (αβα) sandwich, 57 out of 224 under 2-layer sandwich, 6 out of the 18 folds under α/β barrel and 16 out of 58 folds under α/β roll were observed. Among that, some folds were observed in a greater number of proteins compared to other folds (Figure 4). In α class, the Arc Repressor Mutant Subunit A fold and four Helix Bundle fold of bundle architectures were observed in most of the proteins compared to other folds (Table 1). Under β class, the Immunoglobulin- like fold and Jelly Roll fold of β-sandwich, PH-domain fold of β-roll, and OB fold of β-barrel were observed most. Analysis of Long Repeats at the Architecture Level According to CATH domain-based hierarchy (http://www. cathdb.info/browse/tree), the presence of long repeats in diﬀerent architectures of alpha (α), beta (β), and alpha/beta (α/β) class proteins was observed (Figure 3). Out of ﬁve architectures of α class, these were observed in the four architectures, namely orthogonal bundle, up-down bundle, α horseshoe and October 2019 \| Volume 7 \| Article 250 Frontiers in Bioengineering and Biotechnology \| www.frontiersin.org 4 Rajathei et al. Long Repeats in Protein Sequences FIGURE 3 \| Number of Long repeats containing proteins assigned with different architectures of α, β, and α/β class using CATH. barrel and Herpes Virus-1 followed by Alpha-Beta plaits fold of 2-layer Sandwich, and Ubiquitin-like (UB roll) of αβ roll were observed most. The results reveal the predominant occurrence of long repeats in the diverse structure exhibiting folds of the proteins. α/α barrel. Among these, substantial numbers were present in the architectures of bundle and horseshoe. Under β class, the repeats were present in 13 out of 20 architectures and the sandwich, propeller, roll and barrel were observed most. Likewise, repeats were found in 10 out of 14 architectures of α/β class and the architectures of 3-layer (αβα) sandwich, 2-layer sandwich, α/β barrel and αβ-complex were observed most. By combining the architectures from diﬀerent classes of proteins, repeats in speciﬁc architectures of sandwich, bundle, barrel and roll compared to other architectures were found out. October 2019 \| Volume 7 \| Article 250 Frontiers in Bioengineering and Biotechnology \| www.frontiersin.org Analysis of Long Repeats for Intrinsic Disordered Region g The intrinsically disordered regions (IDR) for 51 (<1%) of long repeats proteins were found out using DisProt database. While analyzing the predisposition of long repeats for IDR, most of the repeats were identiﬁed in the structured regions. However, we also identiﬁed long repeats in an IDR. For example, Nucleoporin NUP1 (P20676) protein of length 1,076 contains tandem repeats of length 62 in the region of (352–399/403–462/522–564/666–728/731–778/779–840/849– 906/907–972/978–1,031), which has been identiﬁed as an IDR (300–1,078). This analysis suggests that long repeats are generally structured in most of the proteins while few of them may have IDRs. FIGURE 6 \| Varying larger repeat lengths are observed in the Rossmann fold of the (A) Cobalt chelatase CbiK (2xvyA) with repeat length 103 and (B) Phosphoribosyl pyrophosphate synthetase (3 mbiA) with repeat length 121. The repeats regions are highlighted with different colors. FIGURE 6 \| Varying larger repeat lengths are observed in the Rossmann fold of the (A) Cobalt chelatase CbiK (2xvyA) with repeat length 103 and (B) Phosphoribosyl pyrophosphate synthetase (3 mbiA) with repeat length 121. The repeats regions are highlighted with different colors. Analysis of Long Repeats for Structural Repeats FIGURE 5 \| Long repeats that form structural repeats in the folds of Orthogonal bundle, Up-down bundle of alpha class, Immunoglobulin fold, Jelly Roll, OB fold of beta class, Rossmann fold, Alpha Beta Plait, TIM barrel, and UB roll of alpha-beta class. of βαβαβαβα of length 121 in Figure 6B (Cherney et al., 2011). The analysis results suggest that the length variations of repeats within the Rossmann fold lead to the presence of additional α-helices, β-strands, and coil regions. Thus, longer repeats of diﬀerent lengths provide the structural diﬀerences within a fold of the proteins. FIGURE 6 \| Varying larger repeat lengths are observed in the Rossmann fold of the (A) Cobalt chelatase CbiK (2xvyA) with repeat length 103 and (B) Phosphoribosyl pyrophosphate synthetase (3 mbiA) with repeat length 121. The repeats regions are highlighted with different colors. Analysis of Long Repeats for Structural Repeats The Rossmann fold in 3-layer (αβα) sandwich, TIM Barrel in αβ October 2019 \| Volume 7 \| Article 250 Frontiers in Bioengineering and Biotechnology \| www.frontiersin.org 5 Rajathei et al. Long Repeats in Protein Sequences FIGURE 4 \| The occurrences of certain folds of Arc Repressor Mutant subunit A of Orthogonal bundle architecture, Four helix bundle of Up-down bundle, Jelly Rolls and Immunoglobulin of Beta sandwich, PH-Domain like fold of Beta Roll, OB Roll of Beta Barrel, Rossmann fold of 3-layer sandwich, Alpha Beta plaits, and Herpes Virus-1 domain of 2-layer sandwich, TIM barrel of Alpha-Beta Barrel and UB Roll of Alpha-Beta Roll in a substantial numbers of Long repeats proteins. FIGURE 4 \| The occurrences of certain folds of Arc Repressor Mutant subunit A of Orthogonal bundle architecture, Four helix bundle of Up-down bundle, Jelly Rolls and Immunoglobulin of Beta sandwich, PH-Domain like fold of Beta Roll, OB Roll of Beta Barrel, Rossmann fold of 3-layer sandwich, Alpha Beta plaits, and Herpes Virus-1 domain of 2-layer sandwich, TIM barrel of Alpha-Beta Barrel and UB Roll of Alpha-Beta Roll in a substantial numbers of Long repeats proteins. TABLE 1 \| Number of proteins containing long repeats in the architectures and folds of the proteins. Class Architecture Number of proteins Fold Number of proteins Alpha (α) class Orthogonal bundle 1,272 Arc repressor mutant, subunit A 391 Alpha horseshoe 1,072 Leucine-rich repeats variant 488 Up-down bundle 265 Four helix bundle 32 Beta (β) class Beta sandwich 909 i) Jelly Rolls ii) Immunoglobulin 228 642 7 Propeller 549 Methylamine dehydrogenase 549 Beta roll 334 PH-domain like 175 Beta barrel 234 OB Roll 93 Alpha Beta (αβ) class 3-Layer (aba) Sandwich 1,885 3-layer(αβα) sandwich 1,223 2-Layer sandwich 1,332 Alpha beta plaits 123 Alpha-beta barrel 432 TIM barrel 334 Alpha-beta complex 390 Spore coat polysaccharide biosynthesis protein SpsA 156 Alpha-beta roll 180 UB Roll 32 TABLE 1 \| Number of proteins containing long repeats in the architectures and folds of the proteins. Frontiers in Bioengineering and Biotechnology \| www.frontiersin.org October 2019 \| Volume 7 \| Article 250 Rajathei et al. Long Repeats in Protein Sequences FIGURE 5 \| Long repeats that form structural repeats in the folds of Orthogonal bundle, Up-down bundle of alpha class, Immunoglobulin fold, Jelly Roll, OB fold of beta class, Rossmann fold, Alpha Beta Plait, TIM barrel, and UB roll of alpha-beta class. Analysis of Functions of Long Repeats at the Domain Level Some of the protein families are having long repeats in a greater number of their member proteins (Supplementary Data File 3). Figure 7 shows the list of 36 protein families such as Class II aminoacyl-tRNA synthetase, Ser/Thr Protein kinase, Class I aminoacyl-tRNA synthetase, Cytochrome P450, Mitochondrial carrier (TC 2.A.29), G-protein coupled receptor 1, and ABC transporter that are having repeats in more than 40 member proteins of the family. We observed long repeats in the domains of the family with varying lengths. For example, the Peptidase S8 family proteins contained long repeats in 41 member proteins of the family (Table 2). Among these, 38 protein repeats were in the Peptidase S8 domains with varying repeat lengths. Figure 8 shows some of the proteins’ repeat regions as well as their alignment that covers the Peptidase S8 domain regions. The level of similarity between the repeats in the Peptidase S8 domain within a protein and within the member proteins of the Peptidase S8 domain family was computed in terms of % sequence identity. For example, the sequence identity of 29% was observed for the repeats (157–215/228–313), within the Peptidase domain (157– 401) of the Aqualysin-1 protein (P08594) (Table 2). Further, the sequence similarities of repeat unit (157–215) of this protein, with the repeat units in the Peptidase S8 domain of the 37 member proteins, were also computed. We observed that 65 % of repeats were in the range of 20–40% sequence identity and the remaining protein repeats were in the range of 10–20% identity. This observation suggests that the repeats within a protein, as well as within a protein family, are considerably diverged. Further, repeats in the domains are involved in the function through functional residues (highlighted in red color). For example, the regions (162–173) and (197–207) of repeats (157–215/228–313) of Aqualysin-1 (UniProt ID P08594) have contained functional residues VYVIDTGIRTTH and HGTHVAGTIGG for Serine proteases (Figure 8). The functional involvement of the repeats was also found out in the structure of the proteins using PDBsum. For example, the functionally involved residues (highlighted red in color) of repeats (157–215/228–313) in the structure of Aqualysin-1 (PDB ID 4DZT) were found out using PDBsum search (Figure 9). This suggests that these repeats occur in the domains of the family for the function of the proteins. Analysis of Functions of Long Repeats at the Domain Level The Pfam domain assignments in 26,750 (90%) of proteins were found and suggested the occurrence of repeats in the functional domain families containing proteins. While grouping October 2019 \| Volume 7 \| Article 250 Frontiers in Bioengineering and Biotechnology \| www.frontiersin.org 7 Rajathei et al. Long Repeats in Protein Sequences FIGURE 7 \| List of the 36 protein families that are having long repeats in more than 40 member proteins. an 40 member proteins. FIGURE 7 \| List of the 36 protein families that are having long repeats in more than 40 member proteins. by protein family, the existence of repeats in 5,258 distinct protein families was found out. Some of the protein families are having long repeats in a greater number of their member proteins (Supplementary Data File 3). Figure 7 shows the list of 36 protein families such as Class II aminoacyl-tRNA synthetase, Ser/Thr Protein kinase, Class I aminoacyl-tRNA synthetase, Cytochrome P450, Mitochondrial carrier (TC 2.A.29), G-protein coupled receptor 1, and ABC transporter that are having repeats in more than 40 member proteins of the family. We observed long repeats in the domains of the family with varying lengths. For example, the Peptidase S8 family proteins contained long repeats in 41 member proteins of the family (Table 2). Among these, 38 protein repeats were in the Peptidase S8 domains with varying repeat lengths. Figure 8 shows some of the proteins’ repeat regions as well as their alignment that covers the Peptidase S8 domain regions. The level of similarity between the repeats in the Peptidase S8 domain within a protein and within the member proteins of the Peptidase S8 domain family was computed in terms of % sequence identity. For example, the sequence identity of 29% was observed for the repeats (157–215/228–313), within the Peptidase domain (157– 401) of the Aqualysin-1 protein (P08594) (Table 2). Further, the sequence similarities of repeat unit (157–215) of this protein, with the repeat units in the Peptidase S8 domain of the 37 member proteins, were also computed. We observed that 65 % of repeats were in the range of 20–40% sequence identity and the remaining protein repeats were in the range of 10–20% identity. This observation suggests that the repeats within a protein, as well as within a protein family, are considerably diverged. by protein family, the existence of repeats in 5,258 distinct protein families was found out. Frontiers in Bioengineering and Biotechnology \| www.frontiersin.org Analysis of Enzyme and Non-enzyme Functions of Long Repeats g p Further, the enzyme functions in 13,333 proteins and non- enzyme functions in 2,437 proteins, of a total of 15,770 (53%) of long repeats proteins, were also found out. Of a total of 13,333 enzymes having long repeats, Ligases (35.91%) have the maximum number of repeats followed by Isomerases (28.98%), Translocases (11.81%), Transferases (11.12%), Lyases (5.12%), Hydrolases (4.74%), and Oxidoreductases (2.32%). Among the non-enzymes in 2,437 proteins, NP binding proteins (48.09%) have the maximum number of repeats followed by DNA binding (30.44%), metal binding (16.94%), and calcium binding (4.51%). These observations suggest the importance of long repeats in both the catalytic and binding function of proteins apart from serving as modules of large assemblies. October 2019 \| Volume 7 \| Article 250 Frontiers in Bioengineering and Biotechnology \| www.frontiersin.org 8 Long Repeats in Protein Sequences Rajathei et al. TABLE 2 \| List of 41 member proteins of the Peptidase S8 family long repeat region’s and their function domain regions assigned using Pfam. S. NO. Analysis of Enzyme and Non-enzyme Functions of Long Repeats Protein name (UniProt ID and length) Long repeats regions and their length Peptidase S8 domain regions and their length Other domains and their regions 1 Aqualysin-1 (P08594 514) 157–215/228–313 (57) 157–401 (244) Inhibitor_I9 (54–125) 2 Bacillopeptidase F (P16397 1434) i) 198–279/280–352/355–436/437– 529 (85) ii) 568–609/615–701/1,044– 1,167 (79) 218–504 (286) Peptidase_M6 (667–801) Inhibitor_I9 (68–178) 3 Calcium-dependent protease (Q59149 663) 219–313/315–412 (93) 228–530 (302) P_proprotein (547–662) 4 Cell wall-associated protease (P54423 895) 737–803/818–885 (67) 458–729 (271) 5 Cuticle-degrading protease (P29138 389) 72–171/172–270/277–355 (84) 139–383 (244) Inhibitor_I9 (41–107) 6 Extracellular serine protease (P29805 1046) i) 158–240/241–381/385–491 (137) ii) 509–585/586–685/687–754/771– 831 (83) 71–397 (326) Autotransporter (769–1,045) 7 Microbial serine proteinase (P31339 622) 167–237/389–458 (67) 89–411 (322) P_proprotein (491–572) 8 Minor extracellular protease vpr (P29141 807) 18–148/149–208/340–473/475– 532/658–711 (183) 184–594 (410) Inhibitor_I9 (57–143); PA superfamily (355–497); FlgD_ig superfamily (712–792) 9 Minor extracellular protease Epr (P16396 646) 39–112/169–240/249–327 (74) 137–380 (243) 10 MycP4 protease (I6YC58 456) 25–208/222–407 (159) 86–389 (303) 11 MycP1 protease (A0QNL1 450) 91–172/173–327/332–428 (127) 83–381 (298) 12 Nisin leader peptide-processing serine protease (Q07596 683) 228–281/379–418/504–557 (52) 255–546 (291) 13 PIII-type proteinase (P15292 1963) 156–206/208–209/295–380 (82) 212–698 (486) 14 Proprotein convertase subtilisin/kexin type 9 (Q80W65 695) 467–537/540–611/616–682 (140) 185–423 (238) Inhibitor_I9 (80–152) 15 Pyrolysin (P72186 1399) i) 225–274/276–339/341–400 (62) ii) 959–1,006/1,011–1,158/1,169– 1,296 (126) i) 174–380 (206) ii) 408–654 (246) 16 Putative subtilisin-like proteinase 1 (Q8SQJ3 466) 23–92/94–160/165–195 (67) 144–422 (278) Inhibitor_I (919–90) 17 Putative subtilisin-like proteinase 2(Q8SS86 536) 106–165/278–336/362–390 (60) 272–452 (180) 18 Probable subtilase-type serine protease DR_A0283 (Q9RYM8 729) 84–126/131–209/232–310/320– 378 (78) 183–470 (287) Peptidase_M14NE-CP- C_like(486–558); PPC (624–693) 19 Subtilase-type proteinase psp3 (Q9UTS0 452) 217–283/349–407 (56) 202–429 (227) Inhibitor_I9 (80–162) 20 Subtilase-type proteinase RRT12 (P25381 492) 53–107/269–320 (52) 156–389 (233) 21 Subtilisin-like protease SBT3.13 (Q8GUK4 767) 320–431/608–719 (107) 153–588 (453) Inhibitor_I9 (41–119); PA_Superfamily (384–485) 22 Subtilisin-like protease SBT4.4 (Q9FGU3 742) 65–226/416–581 (150) 137–581 (444) Inhibitor_I9(34–112); PA(338–458) 23 Subtilisin-like protease SBT4.10 (Q9FIM8 694) 138–284/387–534 (139) 138–526 (388) Inhibitor_I9 (35–113);PA (332–371) 24 Subtilisin-like protease SBT4.14 (Q9LLL8 750) 202–333/336–464/467–596 (129) 141–594 (453) Inhibitor_I9 (38–115); PA (346–467) 25 Subtilisin-like protease SBT2.4 (F4HYR6 833) 245–361/362–547/548–736 (178) 169–691 (522) Inhibitor_I9 (70–138);PA (389–533) 26 Subtilisin-like protease SBT4.15 (Q9LZS6 767) 284–379/450–550 (92) 137–590 (453) Inhibitor_I9 (35–113); PA (342–474) 27 Subtilisin-like protease SBT3.18 (Q9STQ2 780) i) 179–224/495–575/707–756 (76) ii) 318–388/405–476 (65) 137–613 (476) Inhibitor_I9 (30–109); PA_Superfamily (361–482) 28 Subtilisin-like protease SBT6.1 (Q0WUG6 1039) 556–644/812–901 (86) 208–486 (278) 29 Subtilisin-like protease SBT2.2 (Q9SUN6 857) 163–222/226–283 (54) 184–674 (490) Inhibitor_I9 (98–159); PA superfamily (406–548) (Continued) region’s and their function domain regions assigned using Pfam. Analysis of Enzyme and Non-enzyme Functions of Long Repeats (Continued) (Continued) October 2019 \| Volume 7 \| Article 250 Frontiers in Bioengineering and Biotechnology \| www.frontiersin.org 9 Long Repeats in Protein Sequences Rajathei et al. TABLE 2 \| Continued S. NO. Protein name (UniProt ID and length) Long repeats regions and their length Peptidase S8 domain regions and their length Other domains and their regions 30 Subtilisin-like protease SBT2.6 (Q9SZV5 817) 155–186/195–224/315–398 (59) 151–635 (484) Inhibitor_I9 (61–124); Pasuperfamily (374–511); fn3_5 superfamily (698–810) 31 Subtilisin-like protease SBT3.6 (Q8L7I2 779) 216–306/307–392 (71) 138–593 (455) Inhibitor_I9 (34–113); PA superfamily (365–493) 32 Subtilisin-like protease SBT1.2 (O64495 776) 150–259/527–633 (101) 127–587 (460) Inhibitor_I9 (27–112); PA superfamily (353–481) 33 Subtilisin-like protease SBT1.4 (Q9LVJ1 778) 169–268/435–532 (86) 133–589 (456) Inhibitor_I9 (32–110); PA superfamily (355–474) 34 Serotype-speciﬁc antigen 1 (P31631 933) 378–433/466–588/594–709 (115) 54–408 (354) Autotransporter superfamily (673–916) 35 Subtilisin-like protease 12 (D4AQA9 417) 253–300/305–372 (64) 145–399 (254) Inhibitor_I9 (35–116) 36 Subtilisin-like protease CPC735_047380 (C5PFR5 401) 84–138/140–196 (53) 143–363 (220) Inhibitor_I9 (35–114) 37 Tripeptidyl-peptidase 2 (Q09541 1375) 370–469/688–782 (87) 89–559 (470) TPPII (832–1,017) 38 Tripeptidyl-peptidase 2 homolog (Q9UT05 1275) i) 265–379/413–522 (96) ii) 637–807/820–958 (121) 90–545 (450) TPPII (837–1,008) 39 Thermophilic serine proteinase (Q45670 402) 121–202/203–282/283–358 (79) 151–392 (241) 40 Tripeptidyl-peptidase 2 (F4JVN6 1381) i) 143–207/343–403/717–760 (62) ii) 1,043–1,188/1,236–1,380 (135) 140–620 (480) TPPII (897–1,078) SMC_N (1,140–1,355) 41 Subtilisin-like protease (Q00139 371) 20–72/80–135 (52) 83-255 (172) P–proprotein (240–370) FIGURE 8 \| The Aqualysin-1(P085594), Bacillopeptidase F(P16397), and Thermophilic serine proteinase (Q45670) protein’s repeats regions and their alignments that are in the Peptidase S8 domain region assigned by Pfam. FIGURE 8 \| The Aqualysin-1(P085594), Bacillopeptidase F(P16397), and Thermophilic serine proteinase (Q45670) protein’s repeats regions and their alignments that are in the Peptidase S8 domain region assigned by Pfam. DISCUSSION of the proteins. Previous studies have focused on structural and functional implications of proteins with homo repeats (Uthayakumar et al., 2012), ﬁbrous repeats (Parry, 2005) and diﬀerent well-characterized repeats of length 5–50 (Andrade et al., 2001). Therefore, an in-depth study of long repeats in Our survey of long repeats in a non-redundant set of UniProt sequences has highlighted the occurrence of these repeats that play an important role in the structure and function of domains October 2019 \| Volume 7 \| Article 250 Frontiers in Bioengineering and Biotechnology \| www.frontiersin.org 10 Long Repeats in Protein Sequences Long Repeats in Protein Sequences Rajathei et al. FIGURE 9 \| The functional residues of repeats (157–215/228–313) in the structural regions (30–88)/(101–186) (highlighted by red and green inverted triangles with red color dots) of Aqualysin-1 proteins are found out using PDBsum search. FIGURE 9 \| The functional residues of repeats (157–215/228–313) in the structural regions (30–88)/(101–186) (highlighted by red and green inverted triangles with red color dots) of Aqualysin-1 proteins are found out using PDBsum search. through repetitive duplication of a small stable unit (βα) (Lang et al., 2000). It has been observed that repeats in the folds may fulﬁll the physical demand (stable and fast folding conformation) of the protein chain during the process of evolution, in order to meet the cellular function (Lupas et al., 2001). Further, it has been shown that the existence of structural symmetries in the super-folds (6 out of 10) may also require larger duplication during evolution of the proteins (Brych et al., 2003). Kim et al. (2010), through their SymD (detecting symmetry in protein structures) method, have identiﬁed 33 folds that contain 10 or more symmetric domains. There is considerable overlap between the symmetry in the folds they identiﬁed and those observed in the present work (Figure 5). We observed that long repeats of diﬀerent lengths within a fold provide the structural diﬀerences of the proteins for diﬀerent functions. Further, the analysis of predisposition of long repeats for disordered regions has shown that long repeat proteins are mostly structured to form stable folds. However, it has been observed that short tandem repeats are highly disordered, which do not adopt a single deﬁned conﬁguration for speciﬁc function (Tompa, 2012; Habchi et al., 2014; van der Lee et al., 2014). Frontiers in Bioengineering and Biotechnology \| www.frontiersin.org CONCLUSIONS the repeats in the individual proteins of PDB and found that the existence of repeats in single/two domains from the same family, for the function of the proteins and that are not in the domains, are also involved in the function of the proteins (Mary Rajathei and Selvaraj, 2013). We observed that the lengths of repeats in the domains of the family are not uniform. Further, the computation of sequence identity of the repeats within a protein and within a family of Peptidase S8 domain shows lower similarity, which may be the consequence of their divergences over a period. Earlier, it was observed that repeat proteins are indeed repetitive in their families, exhibiting abundant stretches of short perfect repetitions (Turjanski et al., 2016). The repeats of varying lengths in the structures of the fold, as well as in the functional domains of the family, have suggested that long repeats are considerably diverged and may not be overlapped. However, further studies would be needed to understand the conservation of long repeats of the proteins in the structure and function of the proteins. The present large scale study has focused on the presence of long repeats in a non-redundant set of the entire annotated UniProtKB/Swiss-Prot database and reveals that long repeats are found in 23% of the proteins. Regarding their three-dimensional structures, they are found in certain structural folds that are incorporated with repetitive duplication of small stable folds. Further, the long repeats of diﬀerent lengths within each fold are observed in diﬀerent structures of the proteins. From a functional perspective, these repeats are found in both enzyme and non- enzyme functions containing proteins. Hence, long repeats may have a role in the evolution of proteins with stable folds and novel functions. SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fbioe. 2019.00250/full#supplementary-material ACKNOWLEDGMENTS DR acknowledges University Grants Commission, India for a research fellowship through UGC Post-doctoral Fellow for Women Research Grant No. F.15-1/2017-18/PDFWM-2017-18- TAM-44286/(SA-II). While commenting on the evolution of the well-characterized short tandem repeats in many evolutionary lineages, it has been postulated that repeat-containing proteins are cheap to evolve, rather than the de nova sequence evolution, as the repeat units are thermodynamically stable (Andrade et al., 2001; Andersson et al., 2015). Through our analysis, we observed the occurrence of long repeats in the stable folds for diﬀerent functions of the proteins and suggested that long repeats may play a role in the evolution of proteins with stable folds and novel functions. DATA AVAILABILITY STATEMENT The UniProt annotated sequence ﬁles and the RADAR output ﬁles were analyzed for this study. Major results are available as Supplementary Material. Further, we observed the existence of long repeats in all seven enzyme classes of the proteins and are especially more abundant in ligases and isomerases. Among the non- enzyme proteins, long repeats are observed in DNA binding, calcium binding, metal binding and NP binding proteins with NP binding and DNA binding in a greater number of proteins. However, further studies are needed to understand why certain enzyme classes and non-enzyme classes are having long repeats in more numbers. This shows that the occurrence of long repeats, not only serves as modules of large assemblies, but also in the catalytic function or binding of the proteins. DISCUSSION UniProt sequences was carried out for a better understanding of the correspondence of repeat sequences with their structures and functions. In this study, we used the RADAR program for internal repeat detection, since it often detects both tandem and interspersed repeats in larger size. Our earlier studies for repeats analysis (Mary Rajathei and Selvaraj, 2013; Mary et al., 2015) have shown the ability of RADAR to detect repeats of length > 50 that are structurally similar and conserved in a 3D structure environment. Further, the sensitivity and accuracy of RADAR repeats, by comparison with Pfam, indicate good coverage, accurate alignments, and reasonable repeat borders (Heger and Holm, 2000). The identiﬁed repeats vary in the range of 50–1,759 of lengths and diverged with more insertions and deletions, but the calculated z-scores by RADAR have shown their statistical signiﬁcance. From a structural perspective, long repeats tend to occur abundantly in certain architectures of sandwich, barrel, bundle, and roll. Within these architectures, they are predominately observed in the super folds of up-down and orthogonal bundle of α-class, Immunoglobulin, Jelly Roll and OB fold of β-class, Rossmann fold, TIM barrel, α/β plait, and UB roll of α/β class of the proteins. The adoption of classic super secondary elements (αα, βαβ, ββ) and incorporation of repetitive duplication of a small stable unit may be the possible reasons for abundance of larger duplication in these folds (Thornton et al., 1999). For example, the evolution of the (βα)8 repeat in the TIM barrel is Further, repeats have been analyzed for a speciﬁc domain of the family, in which protein function could be found out through the domain (Rentzsch and Orengo, 2013). We found that repeats in the domain regions of the family are involved in the function through functional residues. Earlier, we analyzed October 2019 \| Volume 7 \| Article 250 Frontiers in Bioengineering and Biotechnology \| www.frontiersin.org 11 Long Repeats in Protein Sequences Rajathei et al. AUTHOR CONTRIBUTIONS DR developed the computer programs in perl platform for this study and drafted the manuscript. SP supported to analyze and computation the data. SS conceived the idea and helped in the preparation of the manuscript. REFERENCES doi: 10.1073/pnas.89.22.10915 Parry, D. A. (2005). Structural and functional implications of sequence repeats in ﬁbrous proteins. Adv. 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https://openalex.org/W4248670076	https://pure.uva.nl/ws/files/26682127/s41522_018_0054_5.pdf	English	null	Daily Rhythmicity in Coastal Microbial Mats	Springer eBooks	2,021	cc-by	12,012	Citation for published version (APA): Hörnlein, C., Confurius-Guns, V., Stal, L. J., & Bolhuis, H. (2018). Daily rhythmicity in coastal microbial mats. npj Biofilms and Microbiomes, 4, Article 11. https://doi.org/10.1038/s41522- 018-0054-5 UvA-DARE (Digital Academic Repository) Citation for published version (APA): Hörnlein, C., Confurius-Guns, V., Stal, L. J., & Bolhuis, H. (2018). Daily rhythmicity in coastal microbial mats. npj Biofilms and Microbiomes, 4, Article 11. https://doi.org/10.1038/s41522- 018-0054-5 General rights 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), other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons). UvA-DARE (Digital Academic Repository) Daily rhythmicity in coastal microbial mats Hörnlein, C.; Confurius-Guns, V.; Stal, L.J.; Bolhuis, H. DOI 10.1038/s41522-018-0054-5 Publication date 2018 Document Version Final published version Published in npj Biofilms and Microbiomes License CC BY Link to publication Citation for published version (APA): Hörnlein, C., Confurius-Guns, V., Stal, L. J., & Bolhuis, H. (2018). Daily rhythmicity in coastal microbial mats. npj Biofilms and Microbiomes, 4, Article 11. https://doi.org/10.1038/s41522- 018-0054-5 UvA-DARE (Digital Academic Repository) Daily rhythmicity in coastal microbial mats Hörnlein, C.; Confurius-Guns, V.; Stal, L.J.; Bolhuis, H. DOI 10.1038/s41522-018-0054-5 Publication date 2018 Document Version Final published version Published in npj Biofilms and Microbiomes License CC BY Link to publication Citation for published version (APA): Hörnlein, C., Confurius-Guns, V., Stal, L. J., & Bolhuis, H. 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UvA-DARE is a service provided by the library of the University of Amsterdam (https://dare.uva.nl) Download date:24 Oct 2024 www.nature.com/npjbioﬁlms INTRODUCTION the clock is regulated through a bacteriophytochrome, the circadian input kinase A (CikA).13,14 The second abundant group of circadian clock controlled photosynthetic primary producers consists mainly of diatoms.15 However, the underlying mechanism of their circadian clock and its genetic components are not yet fully understood,15 and will not be further discussed here. Phototrophic microbial mats are highly complex and nearly self- sustaining laminated ecosystems that globally develop amongst others in coastal intertidal sandy sediments. The primary producers in microbial mats are oxygenic photoautotrophs, mainly Cyanobacteria and diatoms1 and they supply the community with organic carbon. Many Cyanobacteria also ﬁx atmospheric nitrogen thereby also providing this important nutrient to the community. Phototrophic microbial mats are driven by the daily cycle of light and dark in which processes such as photosynthesis, respiration, fermentation, migration and nitrogen ﬁxation take place at designated times during a 24-h day.2–8 y Circadian clocks, which are ubiquitious among Eukarya, were thought to have evolved outside of this domain only in Cyanobacteria.16–18 However, genome analysis revealed homologs of kaiB and kaiC but not kaiA17 in several other bacterial phyla such as Proteobacteria, Bacteroidetes, Chloroﬂexi and also in Archaea.17,19 Analysis of rhythmicity in gene expression in the Alphaproteobacteria Rhodopseudomonas palustris20 and Rhodo- bacter sphaeroides,21 revealed similar regulatory patterns as in Cyanobacteria lacking kaiA such as in Prochlorococcus marinus.22 Th f k iBC i b h f h Al h b i l d Circadian clocks, which are ubiquitious among Eukarya, were thought to have evolved outside of this domain only in Cyanobacteria.16–18 However, genome analysis revealed homologs of kaiB and kaiC but not kaiA17 in several other bacterial phyla such as Proteobacteria, Bacteroidetes, Chloroﬂexi and also in Archaea.17,19 Analysis of rhythmicity in gene expression in the Alphaproteobacteria Rhodopseudomonas palustris20 and Rhodo- bacter sphaeroides,21 revealed similar regulatory patterns as in Cyanobacteria lacking kaiA such as in Prochlorococcus marinus.22 The presence of kaiBC in both of these Alphaproteobacteria led to higher growth rates and increased ﬁtness under a 24-h light-dark cycle but not under continuous light or dark conditions. ARTICLE OPEN Daily rhythmicity in coastal microbial mats Christine Hörnlein1, Veronique Confurius-Guns1, Lucas J. Stal1,2 and Henk Bolhuis 1 Cyanobacteria are major primary producers in coastal microbial mats and provide biochemical energy, organic carbon, and bound nitrogen to the mat community through oxygenic photosynthesis and dinitrogen ﬁxation. In order to anticipate the speciﬁc requirements to optimize their metabolism and growth during a day-and-night cycle, Cyanobacteria possess a unique molecular timing mechanism known as the circadian clock that is well-studied under laboratory conditions but little is known about its function in a natural complex community. Here, we investigated daily rhythmicity of gene expression in a coastal microbial mat community sampled at 6 time points during a 24-h period. In order to identify diel expressed genes, meta-transcriptome data was ﬁtted to periodic functions. Out of 24,035 conserved gene transcript clusters, approximately 7% revealed a signiﬁcant rhythmic expression pattern. These rhythmic genes were assigned to phototrophic micro-eukaryotes, Cyanobacteria but also to Proteobacteria and Bacteroidetes. Analysis of MG-RAST annotated genes and mRNA recruitment analysis of two cyanobacterial and three proteobacterial microbial mat members conﬁrmed that homologs of the cyanobacterial circadian clock genes were also found in other bacterial members of the microbial mat community. These results suggest that various microbial mat members other than Cyanobacteria have their own molecular clock, which can be entrained by a cocktail of Zeitgebers such as light, temperature or metabolites from neighboring species. Hence, microbial mats can be compared to a complex organism consisting of multiple sub-systems that have to be entrained in a cooperative way such that the corpus functions optimally. ﬁl d b ( ) d / npj Bioﬁlms and Microbiomes (2018) 4:11 ; doi:10.1038/s41522-018-0054-5 Received: 9 November 2017 Revised: 26 March 2018 Accepted: 17 April 2018 1Department of Marine Microbiology and Biogeochemistry, Royal Netherlands Institute for Sea Research, and Utrecht University, Den Hoorn, The Netherlands and 2Department of Freshwater and Marine Ecology, Institute for Biodiversity and Ecosystem Dynamics, University of Amsterdam, Amsterdam, The Netherlands Correspondence: Henk Bolhuis (henk.bolhuis@nioz.nl) 1Department of Marine Microbiology and Biogeochemistry, Royal Netherlands Institute for Sea Research, and Utrecht University, Den Hoorn, The Netherlands and 2Department of Freshwater and Marine Ecology, Institute for Biodiversity and Ecosystem Dynamics, University of Amsterdam, Amsterdam, The Netherlands Correspondence: Henk Bolhuis (henk bolhuis@nioz nl) Published in partnership with Nanyang Technological University Received: 9 November 2017 Revised: 26 March 2018 Accepted: 17 April 2018 INTRODUCTION Entrainment (reset) and, to a certain degree, the output (oscillation period) of Published in partnership with Nanyang Technological University ill h h 24 h l f i id i d i 26 27 14 00 h 1021 d 1528 l 2 1) Th i bi l Fig. 1 Photosynthetically active radiation during the sampling period (A) and peak expression phases of rhythmic CGTs in the metatranscriptome (b). a Photosynthetically active radiation (PAR) (µmol m−2 s−1) measured every minute during the sampling period on the beach of Schiermonnikoog (N = 1429). Stippled lines indicate sampling time of metatranscriptomes (N = 6). b Clustered bar chart plots the peak expression F(h) against the count of rhythmic protein-coding CGTs. CGT-F (forward reads) = black, CGT-R (reverse reads) = gray. The peak expression phase displays the time frame in which gene expression reaches maximum Daily rhythmicity in coastal microbial mats C Hörnlein et al. 2 Daily rhythmicity in coastal microbial mats C Hörnlein et al. 2 Fig. 1 Photosynthetically active radiation during the sampling period (A) and peak expression phases of rhythmic CGTs in the metatranscriptome (b). a Photosynthetically active radiation (PAR) (µmol m−2 s−1) measured every minute during the sampling period on the beach of Schiermonnikoog (N = 1429). Stippled lines indicate sampling time of metatranscriptomes (N = 6). b Clustered bar chart plots the peak expression F(h) against the count of rhythmic protein-coding CGTs. CGT-F (forward reads) = black, CGT-R (reverse reads) = gray. The peak expression phase displays the time frame in which gene expression reaches maximum oscillator through ∼24 h cycles of protein oxidation–reduction.26,27 PRX reacts to a rhythm generation of stress-induced reactive oxygen species (ROS) that are formed in reaction to photosynth- esis during the day and to the reducing-, and anoxic conditions during the night. The central timekeeper of this circadian oscillator is, however, hitherto unknown, but it is assumed to be a basic and most ancient circadian timer in all organisms.27,28 Multiple indications of potential endogenous rhythmicity inducers/ time- keepers in bacterial and archaeal phyla20,21,27,29 led us to the following fundamental research question: Is rhythmic gene expression in phototrophic microbial mats limited to Cyanobac- teria and algae or do other bacteria also exhibit a circadian-like, daily rhythmicity? We used meta-transcriptomics and qPCR to analyze diel rhythmicity in gene expression in six microbial mat samples that were taken at regular time points during a 24-h period. INTRODUCTION This KaiBC based system has been proposed to be part of a ‘proto-circadian’ oscillator that does not persist under constant conditions20 and does not contain other deﬁning circadian characteristics, such as temperature compensation and entrainment.22 This suggests that organisms possessing only kaiB and/or kaiC are limited to hourglass timekeeping (determination of intervals).23,24 Potential temperature compensated, endogeneous circadian rhythms in gene expression have been observed in the human gut bacterium Enterobacter aerogenes25 and may be entrained by its host’s clock- driven signal. Alternatively, the ubiquitous antioxidant peroxir- edoxin (PRX) is highly conserved in all domains of life and has been proposed to function as an alternative, universal circadian The evolution of a circadian clock enabled Cyanobacteria to anticipate the day and night regime by regulating gene expression and protein synthesis accordingly9 providing these organisms with an enhanced ﬁtness relative to mutants lacking a circadian clock.10 The circadian clock is a temperature compen- sated time-keeping mechanism which shows an approximate 24-h (circadian) cycle at constant environmental conditions. The presence of kaiBC in both of these Alphaproteobacteria led to higher growth rates and increased ﬁtness under a 24-h light-dark cycle but not under continuous light or dark conditions. This KaiBC based system has been proposed to be part of a ‘proto-circadian’ oscillator that does not persist under constant conditions20 and does not contain other deﬁning circadian characteristics, such as temperature compensation and entrainment.22 This suggests that organisms possessing only kaiB and/or kaiC are limited to hourglass timekeeping (determination of intervals).23,24 Potential temperature compensated, endogeneous circadian rhythms in gene expression have been observed in the human gut bacterium Enterobacter aerogenes25 and may be entrained by its host’s clock- driven signal. Alternatively, the ubiquitous antioxidant peroxir- edoxin (PRX) is highly conserved in all domains of life and has been proposed to function as an alternative, universal circadian The cyanobacterial circadian clock is encoded by the gene cluster kaiABC and the corresponding proteins regulate the transcription of genes involved in various processes such as photosynthesis and nitrogen ﬁxation.11,12 The phosphorylation state of KaiC acts as a gate keeper to the transcription machinery to which it sends scheduled cues during its 24-h phosphorylation/ dephosphorylation cycle. The rhythmic autokinase activity of the ATPase KaiC enhances through binding of KaiA, while KaiC’s autophosphatase activity is modulated by KaiB. INTRODUCTION 14:00 h: 1021 and 1528 μmol m−2 s−1). The microbial mat was not submersed by seawater during the entire sampling period. Metatranscriptomic analysis of the six mat samples generated between 34,016,641 and 47,247,273 paired-end reads per sample with a total of 247,350,938 high quality reads (Phred: ~38) (Table 1). oscillator through ∼24 h cycles of protein oxidation–reduction.26,27 PRX reacts to a rhythm generation of stress-induced reactive oxygen species (ROS) that are formed in reaction to photosynth- esis during the day and to the reducing-, and anoxic conditions during the night. The central timekeeper of this circadian oscillator is, however, hitherto unknown, but it is assumed to be a basic and most ancient circadian timer in all organisms.27,28 Multiple indications of potential endogenous rhythmicity inducers/ time- keepers in bacterial and archaeal phyla20,21,27,29 led us to the following fundamental research question: Is rhythmic gene expression in phototrophic microbial mats limited to Cyanobac- teria and algae or do other bacteria also exhibit a circadian-like, daily rhythmicity? We used meta-transcriptomics and qPCR to analyze diel rhythmicity in gene expression in six microbial mat samples that were taken at regular time points during a 24-h period. Gene transcript clustering, rhythmicity and annotation Clustering the complete transcriptome dataset in reads of ≥96% sequence identity yielded 24,035 conserved gene transcripts (CGTs), representing 168,239,136 reads (68% of the total dataset) with ~10–16 million reads per sample for the datasets with the forward and reverse reads. Approximately 32% of the reads were either singletons or clustered with less than 120 reads in total and were discarded from further analysis. The remaining datasets were analyzed with the R package metaCycle30 to identify periodicity within gene expression proﬁles. The forward and reverse reads were treated as separate datasets. For the dataset with the forward reads (CGT-F), metaCycle analysis revealed 1875 signiﬁcant (p ≤0.05) rhythmic CGTs representing 5,801,407 reads, 3.4% of the clustered reads and 2.3% of the initial number of reads. Of these rhythmic CGT-Fs, 188 were protein coding. For the reverse dataset (CGT-R), metaCycle analysis revealed 1526 signiﬁcantly rhythmic CGTs that in total represent 3,467,908 reads, ~2.0% of the Published in partnership with Nanyang Technological University RESULTS h h General (MG-RAST) statistics of the 6 metatranscriptomes MG-RAST ID Time of sampling (h) Uploaded sequences (#) Validated reads (%) rRNA features (%) Annotated protein features (%) 4611784.3 02:00 40,792,897 39 78 9 4611785.3 05:00 34,016,641 41 85 6 4604210.3 09:30 41,937,839 33 79 9 4604211.3 14:00 44,490,594 30 82 8 4604212.3 19:30 38,865,694 34 81 8 4604213.3 22:45 47,247,273 35 76 10 Table 1. General (MG-RAST) statistics of the 6 metatranscriptomes MG-RAST ID Time of sampling (h) Uploaded sequences (#) Validated reads (%) rRNA features (%) Annotated protein features (%) 4611784.3 02:00 40,792,897 39 78 9 4611785.3 05:00 34,016,641 41 85 6 4604210.3 09:30 41,937,839 33 79 9 4604211.3 14:00 44,490,594 30 82 8 4604212.3 19:30 38,865,694 34 81 8 4604213.3 22:45 47,247,273 35 76 10 Table 2. Phylogenetic distribution of rhythmic, protein-coding CGTs (#) Domain Phyla Rhythmic CGT (#) Bacteria Cyanobacteria 42 Proteobacteria 8 Firmicutes 4 Fusobacteria 2 Planctomycetes 0 Verrucomicrobia 0 Bacteroidetes 0 Eukarya Stramenopiles 60 Opisthokonta 28 Viridiplantae 25 Rhodophyta 18 Alveolata 5 Euglenozoa 2 Haptophyceae 2 Table 3. Functional annotation of rhythmic, protein-coding CGTs (#) (F + R) Function Rhytmic genes (#) RuBisCO 113 Hypothetical protein 90 Photosystem II 88 Photosystem I 23 tmRNA 17 Chloroplast 14 Transposase 6 ATP synthase 4 COI 4 ITS 4 LSU (rRNA) 4 arfA/Rf2 3 Mitochondrial protein 4 Nitrogen ﬁxation related gene cluster 3 Cytochrome b 2 DNA starvation protein 2 Rnase P subunit RnpB 2 Cold shock protein 1 cox2 1 CRISPR 1 Ferritin 1 High light inducible protein 1 Lipoprotein 1 ncRNA 1 Phycocyanin 1 Plasmid 1 tRNA 1 clustered reads and ~1.4% of the initial number of reads. 205 rhythmic CGT-Rs were protein coding. For each rhythmic gene predicted by metaCycle also the phase was calculated, the time point at which the ﬁtted curve indicates maximal expression. These maxima are not discrete and not necessarily identical to the sampling time points and were therefore grouped around the sampling time points in order to distinguish day (8:00–12:00 and 12:00–16:00) from night (20:00–00:00 and 00:00–4:00) and dusk (04:00–8:00) from dawn (16:00–20:00). The highest number of protein-coding CGTs had their maximum expression between 00:00 h midnight and 04:00 h in the morning (Fig. 1b) followed by a smaller group of CGTs that had their maximal expression between 12:00 and 16:00 h. Published in partnership with Nanyang Technological University MG-RAST annotation and rhythmicity MG-RAST annotation and rhythmicity RESULTS h h Photosynthetically active radiation (PAR) during the full sampling period is presented in Fig. 1a. To cover major changes in light intensity (photon density), two samples were taken at night (02:00 and 22:45 h: 0 μmol m−2 s−1), two at twilight (05:00 and 19:30 h: 2.97 and 169.2 μmol m−2 s−1) and two during the day (09:30 and npj Bioﬁlms and Microbiomes (2018) 11 Published in partnership with Nanyang Technological University Daily rhythmicity in coastal microbial mats C Hörnlein et al. 3 clustered reads and ~1.4% of the initial number of reads. 205 rhythmic CGT-Rs were protein coding. For each rhythmic gene predicted by metaCycle also the phase was calculated, the time point at which the ﬁtted curve indicates maximal expression. These maxima are not discrete and not necessarily identical to the sampling time points and were therefore grouped around the sampling time points in order to distinguish day (8:00–12:00 and 12:00–16:00) from night (20:00–00:00 and 00:00–4:00) and dusk (04:00–8:00) from dawn (16:00–20:00). The highest number of protein-coding CGTs had their maximum expression between 00:00 h midnight and 04:00 h in the morning (Fig. 1b) followed by a smaller group of CGTs that had their maximal expression between 12:00 and 16:00 h. Phylogenetic annotation of rhythmic CGTs revealed approxi- mately one third without a blast hit against the NCBI NR (protein) or NT (nucleotide) database. Reads with a blast hit were divided 3) that were maximally expressed between 00:00 and 04:00 h Table 2. Phylogenetic distribution of rhythmic, protein-coding CGTs (#) Domain Phyla Rhythmic CGT (#) Bacteria Cyanobacteria 42 Proteobacteria 8 Firmicutes 4 Fusobacteria 2 Planctomycetes 0 Verrucomicrobia 0 Bacteroidetes 0 Eukarya Stramenopiles 60 Opisthokonta 28 Viridiplantae 25 Rhodophyta 18 Alveolata 5 Euglenozoa 2 Haptophyceae 2 Table 3. Functional annotation of rhythmic, protein-coding CGTs (#) (F + R) Function Rhytmic genes (#) RuBisCO 113 Hypothetical protein 90 Photosystem II 88 Photosystem I 23 tmRNA 17 Chloroplast 14 Transposase 6 ATP synthase 4 COI 4 ITS 4 LSU (rRNA) 4 arfA/Rf2 3 Mitochondrial protein 4 Nitrogen ﬁxation related gene cluster 3 Cytochrome b 2 DNA starvation protein 2 Rnase P subunit RnpB 2 Cold shock protein 1 cox2 1 CRISPR 1 Ferritin 1 High light inducible protein 1 Lipoprotein 1 ncRNA 1 Phycocyanin 1 Plasmid 1 tRNA 1 Table 1. RESULTS h h Phylogenetic annotation of rhythmic CGTs revealed approxi- mately one third without a blast hit against the NCBI NR (protein) or NT (nucleotide) database. Reads with a blast hit were divided into ribosomal RNA (rRNA)-coding hits (mostly of micro-eukaryote origin), hits to whole genomes without speciﬁc annotation, and hits to mainly protein-coding genes. The number of protein- coding CGTs for the forward and the reverse datasets varied respectively between 66 and 138 (188 unique CGTs in total) and between 142 and 116 (205 unique CGTs in total) when blasted against the protein or nucleotide dataset, respectively. The majority of protein-coding genes in each dataset was derived from algae belonging to the phyla of Stramenopiles, Opisthokonta and Viridiplantae, while approximately one fourth was attributed to Bacteria, mainly Cyanobacteria and Proteobacteria (Table 2). Functional annotation revealed several genes encoding the large (rbcL) and small (rbcS) subunits of ribulose-1.5-bisphosphate carboxylase/oxygenase (the CO2-ﬁxing enzyme RuBisCO) (Table 3) that were maximally expressed between 00:00 and 04:00 h (Table S1). Photosystem I (e.g., psaA) and II (e.g., psbA) genes were abundant amongst the protein annotated rhythmic reads (Table 3 and S1) and were maximally expressed between 00:00 and 04:00 h. Several hypothetical protein coding genes had their maximal expression either at night or in the afternoon. 3) that were maximally expressed between 00:00 and 04:00 h (Table S1). Photosystem I (e.g., psaA) and II (e.g., psbA) genes were abundant amongst the protein annotated rhythmic reads (Table 3 and S1) and were maximally expressed between 00:00 and 04:00 h. Several hypothetical protein coding genes had their maximal expression either at night or in the afternoon. MG-RAST annotation and rhythmicity The vast majority of rhythmic genes, 275 and 182, revealed peak expressions between 04:00 and 08:00 h, and between 08:00 and 12:00 h respectively (Fig. 2b). The genes encoded mostly for proteins involved in carbohydrate turn-over, amino acid metabolism, protein biosynthesis and biodegradation, photosynthesis (i.e., photosystem II proteins psbCDJX) and respiration (between 04:00 and 08:00 h only) (Table S3). Recruit- ment to the genome of the cyanobacterium Coleofasciculus (Microcoleus) chthonoplastes PCC7420 (CCY9604) revealed 105 rhythmic genes out of 8109 CDS (Table 4), the majority of which revealed highest expression between 00:00 and 04:00 h (30 RGs) and between 08:00 and 12:00 h (31 RGs). More than 50% of the rhythmic genes encoded hypothetical proteins while others were involved in nitrogen ﬁxation (00:00h–04:00 h) or stress response (00:00h–04:00, 08:00h–12:00 h). ( , ) Recruitment against the genomes of three proteobacterial species, the alphaproteobacterium Roseobacter denitriﬁcans OCh114 and the gammaproteobacteria Allochromatium vinosum DSM180 and Congregibacter litoralis KT71, revealed the highest number of rhythmic genes transcribed during the night between 00:00 and 04:00 h (Fig. 2b), while no rhythmic genes were detected with peak expression between 12:00 and 20:00 h (R. denitriﬁcans, A. vinosum) or 12:00 and 16:00 h (C. litoralis). As presented in Table 4 and Table S3, R. denitriﬁcans revealed 81 rhythmic genes out of 4007 CDS, encoding proteins involved in protein metabolism, carbohydrate turn-over and photosynthesis. A. vinosum revealed 44 rhythmic genes out of 3883 CDS, encoding proteins involved in amino acid biosynthesis, carbohydrate turn- over and respiration. C. litoralis revealed 35 rhythmic genes out of 3220 CDS, encoding predominantly proteins involved in protein metabolism, photosynthesis and stress response. function could be assigned. The majority of rRNA reads was of eukaryal origin (96%) leaving only 3% of bacterial rRNA reads conﬁrming the efﬁcient bacterial rRNA removal. Of the protein- coding genes, 69% was of bacterial and 31% of eukaryal origin. The bacterial protein-coding reads were assigned to Cyanobac- teria, Proteobacteria, and Bacteroidetes while the eukaryal part belonged mainly to Bacillariophyceae. function could be assigned. The majority of rRNA reads was of eukaryal origin (96%) leaving only 3% of bacterial rRNA reads conﬁrming the efﬁcient bacterial rRNA removal. Of the protein- coding genes, 69% was of bacterial and 31% of eukaryal origin. The bacterial protein-coding reads were assigned to Cyanobac- teria, Proteobacteria, and Bacteroidetes while the eukaryal part belonged mainly to Bacillariophyceae. MG-RAST annotation and rhythmicity One third of the raw reads submitted to the MG-RAST metagenome server matched to their pair and passed the quality control which includes having a minimum size requirement of 75 nt for analysis. On average 80% (4,011,978) of these reads were annotated as rRNA, ~8% (428,087) were identiﬁed as protein- coding with known functions while for ~11% (561,507) no Published in partnership with Nanyang Technological University npj Bioﬁlms and Microbiomes (2018) 11 Daily rhythmicity in coastal microbial mats C Hörnlein et al. 4 subsystem proteins or as protein- and DNA metabolism proteins (Table S3). 25% (23 RGs) were maximally expressed between 12:00 and 16:00 h and were mostly annotated as clustering-based subsystem proteins and vitamin and pigment biosynthesis proteins (e.g., tetrapyrrole). The eukaryal rhythmic genes, which were most highly expressed in the early morning, mainly encoded proteins involved in protein metabolism, photosynthesis and respiration (Table S3). Table 4. (A) Number of total rhythmic genes of forward (F) and reverse (R) reads of protein-coding CGTs and (B) of dataset B-C, P, Bs, C and Eu. (C) Number of rhythmic genes obtained by recruitment analysis of L. aestuarii PCC8106, C. chthonoplastes CCY9604, C. litoralis KT71, R. denitriﬁcans Och114 and A. vinosum DSM180. Displayed numbers where ﬁltered for 20–24 h cycle periods and p ≤0.05/0.01. P = B-C and BS = B-C displays amount of shared rhythmic genes between the P/Bs and B-C dataset. The fractions of the total CGTs/genes are given Database/ reference genome Rhythmic genes (#) p ≤0.05 Rhythmic genes (#) p ≤0.01 % of initial CGTs/genes A CGT-Fa 188 79 0.78 CGT-Ra 205 8 0.85 B B-C 265 115 8.5 P 220 109 7.1 Bs 119 47 8.2 C 91 32 5.9 Eu 36 13 5.4 P = B-C 137 Bs = B-C 48 C L. aestuarii 613 203 10 C. chthonoplastes 105 23 1.6 R. denitriﬁcans 81 5 9 A. vinosum 44 2 2 C. litoralis 35 4 5.3 aProtein-coding Rhythmicity in recruited genomes Rhythmicity in recruited genomes Figure 2b depicts the distribution of the peak expression of rhythmic genes, recruited using ﬁve different reference genomes. Recruitment against the genome of the cyanobacterium Lyngbya aestuarii PCC8106 (synonym = Lyngbya sp. CCY9616) revealed 613 rhythmic genes out of a total of 5811 coding sequences (CDS) (Table 4). MG-RAST annotation and rhythmicity g y p y To get a better insight in the taxonomic distribution of rhythmically expressed genes, the genes and their normalized abundance were assigned by the MG-RAST analysis pipeline and divided in the following taxonomic subsets: B-C (bacterial reads excluding cyanobacterial reads), P (proteobacterial reads), Bs (bacteroidetal reads), C (cyanobacterial reads) and Eu (eukaryal reads). The highest number of rhythmic genes (RGs) was found in the bacterial dataset B-C (265 RGs), followed by datasets P (220 RGs), Bs (119 RGs), C (91 RGs) and Eu (36 RGs) (Table 4, Figure S1). About 39% of rhythmic genes found in dataset B-C also occurred in P whereas 12% matched RGs found in Bs. The combined datasets, revealed between 60 and 80% of the rhythmic genes maximally expressed during the early morning between 00:00 and 04:00 h (210 (B-C); 166 (P); 94 (Bs); 27 (C); 23 (Eu) RGs) (Fig. 2a, Table S3). The rhythmic genes of dataset B-C and P that contributed to the highest peak (Fig. 2a) encoded mainly proteins involved in vitamin and pigment biosynthesis (e.g., tetrapyrrole), clustering-based subsystem proteins (i.e., proteins with conﬁrmed functional coupling but of unknown function) and carbohydrate turn-over (e.g., fermentation (B-C), CO2 ﬁxation (B-C, P) and (poly-) saccharide utilization (B-C, P) (Table S3). A major part of Bs-derived rhythmic genes, expressed during the early morning, encoded proteins involved in clustering-based subsystems and protein metabolism (e.g., ribosomal large subunit (LSU) and small subunit (SSU)). An attempt to recruit specimen of Bacteroidetes failed, due to an insufﬁcient amount of sequences. Nitrogenase activity, nifH, and psbA expression Circadian control of a number of cyanobacterial genes is well established and includes genes involved in nitrogen ﬁxation and photosynthesis. Total nitrogenase activity (acetylene reduction) in natural microbial mat samples, derived from the same sampling site as the metatranscriptomes, is depicted in Fig. 3a. The overall activity was low during the night (21:30−3:30 h) reached maximum in the morning (9:30 h) becoming minimal at dusk (21:30 h). The amount of chlorophyll a-normalized ethylene production varied between 1.1 and 5.9 µmol mg−1 h−1. In order to quantify nifH and psbA transcripts from L. aestuarii PCC8106 in the microbial mat samples we estimated their relative expression obtained by normalization against two housekeeping genes (HKG), by means of reverse transcriptase quantitative PCR (RT- qPCR). npj Bioﬁlms and Microbiomes (2018) 11 Published in partnership with Nanyang Technological University MG-RAST annotation and rhythmicity Validation of rnpA and ppc, using BestKeeper, ranked ppc (r = 0.914, p = 0.001) before rnpA (r = 0.843, p = 0.001) as the most stable and evaluated both as suitable HKGs (Table S2). Relative expression of nifH from L. aestuarii was highest during the night Thirty percent of the cyanobacterial RGs (29 RGs) was maximally expressed between 00:00 and 04:00 h of which almost half (12 RGs) of the encoded proteins annotated as clustering-based Published in partnership with Nanyang Technological University Daily rhythmicity in coastal microbial mats C Hörnlein et al. ak expression phase distribution (metaCycle) of the rhythmic genes of the MG-RAST datasets (a) and of the recruited genomes bar charts in a and b plot the peak expression phase (h) distribution of rhythmic genes against their count (#). Bar coloration t (white) and dark (gray) sampling times y y y C Hörnlein et al. 5 Fig. 2 a, b Peak expression phase distribution (metaCycle) of the rhythmic genes of the MG-RAST datasets (a) and of the recruited genomes (b). Clustered bar charts in a and b plot the peak expression phase (h) distribution of rhythmic genes against their count (#). Bar coloration indicates light (white) and dark (gray) sampling times order Bangiales and speciﬁcally the genus Porphyra (~14.6%) (Table S5). Both L. aestuarii recruited psbA transcripts and the MG- RAST annotated psbA transcripts reached a maximum expression at 14:00 h (Fig. 3c,e), while being signiﬁcantly lowest expressed (p = 0.0038) in L. aestuarii at 05:00 h (Fig. 3c). and early morning (02:00, 05:00 h) (Fig. 3b). At 19:30 h nifH was signiﬁcantly lowest expressed (p = 0.0076) (Fig. 3b). The program metaCycle estimated the relative expression of nifH of L. aestuarii as signiﬁcantly rhythmic (p = 0.037) and predicted its peak expression at ~ 03:00 h (Table S4). MG-RAST annotated cyanobac- terial nifH transcripts were derived from unicellular Cyanobacteria of the order Chroococcales (mainly Cyanothece) (~48.5%) and ﬁlamentous Cyanobacteria of the order Oscillatoriales (Trichodes- mium) (~27%) (Table S5). The majority of proteobacterial annotated nifH transcripts was derived from Deltaproteobacteria (i.e., Geobacter, Pelobacter, Rhodospirillum) (~10.5%) (Table S5). The pooled MG-RAST nifH expression was determined as highly signiﬁcantly rhythmic (p = 0.003) with an estimated maximum expression around 12:00 h (Table S4). Published in partnership with Nanyang Technological University MG-RAST annotation and rhythmicity The MG-RAST derived based dataset displayed a maximum in nifH expression dissimilar to the observed maximum in nitrogenase activity with a major peak in the night (02:00 h) and a second minor peak in the evening (19:30 h) (Fig. 3d). and early morning (02:00, 05:00 h) (Fig. 3b). At 19:30 h nifH was signiﬁcantly lowest expressed (p = 0.0076) (Fig. 3b). The program metaCycle estimated the relative expression of nifH of L. aestuarii as signiﬁcantly rhythmic (p = 0.037) and predicted its peak expression at ~ 03:00 h (Table S4). MG-RAST annotated cyanobac- terial nifH transcripts were derived from unicellular Cyanobacteria of the order Chroococcales (mainly Cyanothece) (~48.5%) and ﬁlamentous Cyanobacteria of the order Oscillatoriales (Trichodes- mium) (~27%) (Table S5). The majority of proteobacterial annotated nifH transcripts was derived from Deltaproteobacteria (i.e., Geobacter, Pelobacter, Rhodospirillum) (~10.5%) (Table S5). The pooled MG-RAST nifH expression was determined as highly signiﬁcantly rhythmic (p = 0.003) with an estimated maximum expression around 12:00 h (Table S4). The MG-RAST derived based dataset displayed a maximum in nifH expression dissimilar to the observed maximum in nitrogenase activity with a major peak in the night (02:00 h) and a second minor peak in the evening (19:30 h) (Fig. 3d). Expression of circadian clock (related) genes and peroxiredoxin In order to focus on the circadian clock genes, the relative expression of the cyanobacterial circadian clock core genes kaiA, kaiB, kaiC and of the circadian input kinase cikA were followed by qPCR and compared to the MG-RAST annotated homologs. In addition, the expression proﬁles from the conserved circadian marker peroxiredoxin (prx) were also investigated. Relative expression was obtained by normalizing target gene transcript concentrations (copies/µl) to the geometric mean of the house- keeping gene transcript concentrations. Figure 4 shows the relative expression of L. aestuarii PCC8106 kaiA, kaiB, kaiC, cikA and prx (Fig. 4a) and their MG-RAST abundance in the metatranscriptomes (Fig. 4b). Relative expression of L. aestuarii kaiA, kaiB and kaiC peaked in the morning (9:30 h) and declined in The majority of the MG-RAST annotated psbA transcripts was derived from the cyanobacterial order Chroococcales (Cyanothece, Synechococcus) (~59%) while ~21% originated from the red algae Published in partnership with Nanyang Technological University npj Bioﬁlms and Microbiomes (2018) 11 Daily rhythmicity in coastal microbial mats C Hörnlein et al. Fig. MG-RAST annotation and rhythmicity 3 a–e Nitrogenase activity (a), nifH expression proﬁles (b, d) and psbA expression proﬁles (c, e) of MG-RAST and qPCR. Boxplots display (a the nitrogenase activity (NA) (µmol C2H4 (µg Chl a * h)−1) and relative expression (qPCR) of (b) nifH and (c) psbA in L. aestuarii of which th biological triplicates were taken at 8 (NA) and 6 (relative expression) time points within 24 h. ANOVA results are indicated by asterisk displaying signiﬁcant differences (p < 0.05) in gene expression between time points based on their deviation from the mean (dotted line). Th line charts show the MG-RAST abundance of (d) nifH in Cyanobacteria (black triangle) and Proteobacteria (gray circle), and (e) psbA Cyanobacteria (black triangle) and Bangiophyceae (gray diamond). White and black bars on top of charts indicate light and dark periods. Erro bars display standard deviations (SD) of biological triplicates C Hörnlein et al. 6 3 a–e Nitrogenase activity (a), nifH expression proﬁles 1 Fig. 3 a–e Nitrogenase activity (a), nifH expression proﬁles (b, d) and psbA expression proﬁles (c, e) of MG-RAST and qPCR. Boxplots display (a) the nitrogenase activity (NA) (µmol C2H4 (µg Chl a * h)−1) and relative expression (qPCR) of (b) nifH and (c) psbA in L. aestuarii of which the biological triplicates were taken at 8 (NA) and 6 (relative expression) time points within 24 h. ANOVA results are indicated by asterisks displaying signiﬁcant differences (p < 0.05) in gene expression between time points based on their deviation from the mean (dotted line). The line charts show the MG-RAST abundance of (d) nifH in Cyanobacteria (black triangle) and Proteobacteria (gray circle), and (e) psbA in Cyanobacteria (black triangle) and Bangiophyceae (gray diamond). White and black bars on top of charts indicate light and dark periods. Error bars display standard deviations (SD) of biological triplicates the evening (19:30 or 22:45 h (kaiC)) (Fig. 4a). The highest relative expression of L. aestuarii cikA was at night (2:00 h) and the lowest in the evening (19:30 h) (Fig. 4a). L. aestuarii kaiA and cikA expression proﬁles were signiﬁcantly rhythmic (p = 0.03/0.01) and had their peak expression between 00:00 and 04:00 h and 04:00 and 08:00 h respectively (Table S4). Published in partnership with Nanyang Technological University npj Bioﬁlms and Microbiomes (2018) 11 MG-RAST annotation and rhythmicity The small pool of MG-RAST annotated kaiA transcripts (MG-RAST abundance = 2) of the metatranscriptomes was only detected in the night (2:00 h), while kaiB transcripts (MG-RAST abundance = 24) were most abundant in the early evening (19:30 h) and at dusk (22:45 h) (Fig. 4a). Transcripts of kaiC (MG-RAST abundance = 42) peaked at noon (14:00 h) and at night (2:00 h) (Fig. 4b) while cikA transcripts (MG- RAST abundance = 34) displayed the highest abundance in the beginning of the night (22:45 h). Synechococcus) (~42%), Nostocales (Nostoc) (~27%) and Oscillator- iales (Trichodesmium) (~23%) (Table S4). The majority of the kaiC transcripts also originated from Chroococcales (Chrocosphaera, Cyanothece, Synechococcus) (~56%) and Oscillatoriales (Trichodes- mium) (~22%) (Table S4). Although the majority of clock genes was derived from Cyanobacteria, about 2–3% of the kaiB and kaiC transcripts was derived from archaeal Methanomicrobiales (kaiB and kaiC) and from the bacterial orders Flavobactoriales (kaiB) and Myxococcales (kaiC) (Table S4). The majority of cikA transcripts was also of cyanobacterial origin, namely of the orders Chroococcales (Cyanothece) (~64%) and Nostocales (Nostoc) (~28%). However, ~6% of the cikA annotated genes was derived from Rhizobiales (Rhodopseudomonas, Methylobacterium) (Table S5). The expression of L. aestuarii prx gene peaked in the morning (5:00, 9:30 h) and dropped at dusk (22:45 h) (Fig. 4a). MG-RAST annotated prx transcripts were traced back to mainly Bacter- oidetes of the order Flavobacteriales (Zunongwangia) (~87% of The majority of kaiA sequences in the MG-RAST dataset was derived from Cyanothece, while kaiB transcripts were derived from the cyanobacterial orders Chroococcales (Cyanothece, Published in partnership with Nanyang Technological University Daily rhythmicity in coastal microbial mats C Hörnlein et al. scripts), of which peak expressions preceded those of L. ii (Fi 4 ) b b t 3 4 h Th i ﬁl f th as well as in Archaea. To resolve this question, this study use bi d t t i t i d PCR b d 4 Expression proﬁles of cikA, kaiABC and prx of L. aestuarii PCC8106 (CCY9616) (a) and of the MG-RAST metatranscriptomes (b). a w the relative expression (normalized to the housekeeping genes rnpA and ppc) of the L. MG-RAST annotation and rhythmicity aestuarii circadian clock core genes kaiABC dian input kinase cikA and peroxiredoxin (prx) and (b) the MG-RAST abundance of the summed expression of the aforementioned ge es show the 6 sampling times within the 24 h period while the white and gray coloration of bars symbolize light and dark periods. E display standard deviations (SD) of biological triplicates 7 Fig. 4 Expression proﬁles of cikA, kaiABC and prx of L. aestuarii PCC8106 (CCY9616) (a) and of the MG-RAST metatranscriptomes (b). a Bars show the relative expression (normalized to the housekeeping genes rnpA and ppc) of the L. aestuarii circadian clock core genes kaiABC, the circadian input kinase cikA and peroxiredoxin (prx) and (b) the MG-RAST abundance of the summed expression of the aforementioned genes. X-axes show the 6 sampling times within the 24 h period while the white and gray coloration of bars symbolize light and dark periods. Error bars display standard deviations (SD) of biological triplicates transcripts), of which peak expressions preceded those of L. aestuarii (Fig. 4a) by about 3–4 h. The prx expression proﬁles of the L. aestuarii qPCR, recruitment and MG-RAST data were not signiﬁcantly rhythmic. as well as in Archaea. To resolve this question, this study used a combined metatranscriptomics- and qPCR based gene quantita- tion approach to study the daily rhythm in gene expression in a microbial mat community. Previous metagenomic analysis of coastal microbial mats revealed a low percentage (1–5%) of reads originating from Eukarya.31–33 As expected, we found rhythmic genes belonging to Cyanobacteria as well as those belonging to diatoms (Bacillar- iophyceae). Similar to Cyanobacteria and multicellular Eukarya, circadian control of metabolic processes in oxygenic photosyn- thetic micro-eukaryotes may provide a ﬁtness advantage when anticipating the day/night regime.34–36 The mechanism of the circadian clock of diatoms is poorly understood and, hence, the identiﬁcation of potential clock genes has been proven difﬁ- cult.37,38 Rhytmic gene expression in diatomes may also be inﬂuenced by the tides,7,39 which would inﬂuence endogenously Published in partnership with Nanyang Technological University npj Bioﬁlms and Microbiomes (2018) 11 DISCUSSION Many organisms synchronize their metabolism with the Earth’s rotation cycle. However, apart from the domain Eukarya, it was thought that only Cyanobacteria possess a circadian clock. However, it is difﬁcult to understand why also not other Bacteria or Archaea developed a time-keeping mechanism, while this is obviously an important property. The occurrence of key cyano- bacterial circadian clock genes in other Bacteria17,19 and the occurrence of the circadian peroxiredoxin redox-cycles,27 may hint to a circadian-like rhythmicity in Bacteria other than Cyanobacteria npj Bioﬁlms and Microbiomes (2018) 11 Published in partnership with Nanyang Technological University Daily rhythmicity in coastal microbial mats C Hörnlein et al. 8 induced cycles. Nevertheless, the substantial contribution of diatoms to the number of rhythmic CGTs, which was also reﬂected in the MG-RAST analyzed rRNA pool, showed that these micro- eukaryotes played an important role in the observed daily rhythmicity of gene expression in the microbial mat. also yielded twice as many rhythmic genes for Roseobacter denitriﬁcans when compared to C. litoralis or to A. vinosum. Furthermore, R. denitriﬁcans displayed a higher fraction of rhythmic genes in its genome than the cyanobacterium C. chthonoplastes. The rhythmic transcription of genes involved in carbohydrate turnover, fermentation and polysaccharide utiliza- tion during the night in organisms other than Cyanobacteria may hint to the tight coupling of gene transcription to the availability of substrates produced by the phototrophic organisms during the day. A similar tight coupling of gene expression was observed for animal gut microbiomes that displayed daily rhythmicity of gene expression in response to the host feeding schedule.25,55 Gasol et al.56 suggested that a day–night cycle in Bacteria implies a tight coupling of the photosynthetically produced dissolved organic carbon and its subsequent consumption. This is in agreement with our results and also conﬁrms the results of Poretsky et al.54 and Ottesen et al.22 y y g p The majority of rhythmic genes in algae and Cyanobacteria encoded proteins involved in CO2 ﬁxation and oxygenic photo- synthesis, two processes that are well-known to be under circadian clock control.40–42 The RuBisCO genes rbcL and rbcS and the photosystem II-coding gene psbA dominated the rhythmic CGT dataset, of which the majority was derived from oxygenic photoautotrophs. DISCUSSION However, our experiments conﬁrm the expression of cyanobacterial kai-genes, bacterial kai-homologs (e.g., A. vinosum) and prx genes (e.g., R. denitriﬁcans and C. litoralis) and indicate the presence of potentially circadian clock controlled organisms that may have contributed to the observed rhythmicity in gene expression. Rhythmic analysis uncovered 2–10% rhythmic genes in C. chthonoplastes and L. aestuarii, a number far lower than found in the promoter trap studies in Synechococcus elongates that predicted global circadian control of nearly all genes investi- gated.47 However, it must be taken into account that results of rhythmicity obtained from homogenously grown monocultures in laboratory experiments may not necessarily reﬂect what happens in a multispecies heterogeneous environment such as the microbial mat. However, a microarray study of Synechocystis sp. PCC 6803 also showed only up to 9% of the genes under circadian clock control,48 which is in line with our study. A discrepancy between the amount of rhythmic genes based on mRNA vs. promoter studies was also seen in the eukaryote Arabidopsis,49,50 and was attributed to post-transcriptional control mechanisms that keep the abundance of some mRNAs constant thereby counteracting their rhythmic transcriptional activity.51 Even at constant transcription and protein expression rates, the actual activity can be rhythmically regulated as was shown for RuBisCO expression in the marine alga Gonyaulax sp.52 Here RuBisCO activity depended on the actual distribution of the enzyme within the chloroplast and was causally related to the rhythmic ﬁxation of carbon dioxide. Another essential discovery made in our study is that rhythmic gene expression was not restricted to Cyanobacteria and micro- eukaryotes. Notably, more than 50% of the rhythmic genes were derived from Proteobacteria putting this phylum in the spotlight for future studies of daily rhythms. Key to the (genome-wide) control of metabolic processes by a daily rhythm is the presence of potential Zeitgeber.20,53 In microbial mats, rhythmic control mechanisms such as the circadian clock genes kaiABC and the universal oscillator prx were not conﬁned to Cyanobacteria. Approximately 2 to 4% of kaiB and kaiC transcripts in the MG- RAST dataset were assigned to Bacteroidetes, Proteobacteria and to Euryarchaeota. Furthermore, the majority of prx transcripts was derived from Bacteroidetes. Proteobacteria, and to a lesser extent Bacteroidetes, expressed several rhythmic genes with a periodicity of approximately 24 h. DISCUSSION Expression of the RuBisCO genes and psbA was maximal at low light, between midnight and early morning in accordance with laboratory studies of cultures of Synechococcus elongatus.43 Nitrogen ﬁxation in non-heterocystous Cyanobacteria is often conﬁned to the dark or to low light conditions44,45 and this was also found in our study, concomitantly with nifH expression in L. aestuarii. nifH expression proﬁles of MG- RAST and of L. aestuarii were both signiﬁcantly rhythmic. Unexpectedly, expression of psbA in the MG-RAST dataset and recruitment- and qPCR data was not signiﬁcantly rhythmic. However, arhythmically expressed genes can still lead to rhythmic protein expression (e.g., rpoD4)43 and vice versa (e.g., kaiA) due to post-transcriptional control.46 Several phototrophic microorganisms such as diatoms and Cyanobacteria show diel vertical migration patterns in microbial mats. Migration to deeper, darker and low oxygen parts of a microbial mat protect cells against high UV radiation and inhibiting oxygen concentrations during the day and allow Cyanobacteria to migrate up to 200 µm in a mat57,58 while at night sulﬁde may accumulate to toxic concentrations result in a migration back to the surface.3 Migration patterns in diatoms in marine sediment are linked to both the diel light cycle and to the tidal cycle with an initial endogenous controlled mechanism followed by a physiological response.59 In Cyanobacteria, it has not yet been established whether tidal and circadian control contribute to the cyanobacterial migration,60 or alternatively whether migration through the different micro-gradients in the mats itself affect gene expression. Our analysis revealed rhythmic expressed ﬂagellar protein coding genes in Proteobacteria (ﬂiG, ﬂaA), this may suggest rhythmic motility but not necessarily vertical migration while benthic Cyanobacteria and diatoms lack ﬂagella and migrate through gliding motility, which speed and direction is mainly controlled by light.61,62 The sampling depth of ~5 mm as was applied in this study covers the effect of migration. Manipulation of the natural microbial mat ecosystem would have led to complex unknown environmental effects, which would have made the interpretation difﬁcult or even impossible. Therefore, we decided not to study gene expression while putting the microbial mat under constant dark or light conditions (the latter would have raised questions about the light source and -intensity and would have been impractical in the ﬁeld). Hence, we were unable to determine with certainty that the observed rhythmicity in the microbial mat was caused by an endogenous Zeitgeber rather than by environmental cues. Published in partnership with Nanyang Technological University npj Bioﬁlms and Microbiomes (2018) 11 RNA extraction, sequencing and RT-qPCR and algae are not the only organisms in a microbial mat that may possess a circadian clock. The wide spread occurrence of kaiB and kaiC homologs, the proposed universal circadian oscillator gene prx, and the experimental evidence for light-entrained rhythmic expression among several Proteobacteria is strong evidence for the presence of a circadian clock in other microorganisms.20,21 Nevertheless, it is too early to conclude that all microbial mat members possess a clock. Many members of the microbial mat community are still unknown but the prx gene might be a good candidate universal oscillator. The nature of our experimental setup using a natural system under natural conditions, does not allow conclusive proof that light and temperature were the Zeitgebers. However, it is nevertheless likely that these factors entrained the daily gene expression in several mat members as the Cyanobacteria, eukaryotic algae and the Alphaproteobac- teria.20,21 Rhythmic release of organic molecules such as photo- synthate (mainly polysaccharides) during the day and fermentation products such as low molecular weight organic acids (lactate, acetate and ethanol)63 and nitrogen-containing organic compounds during the night64 may serve as additional Zeitgebers. Regulation of gene expression by external stimuli and ﬂuctuating concentrations of substrate is well documented in Bacteria,65,66 while rhythmic synchronization through quorum- sensing, as was shown for Vibrio ﬁscheri,67 could also contribute to gene expression in this complex high diverse and biomass community.68 and algae are not the only organisms in a microbial mat that may possess a circadian clock. The wide spread occurrence of kaiB and kaiC homologs, the proposed universal circadian oscillator gene prx, and the experimental evidence for light-entrained rhythmic expression among several Proteobacteria is strong evidence for the presence of a circadian clock in other microorganisms.20,21 , q g q RNA was isolated from each sample using the RNA PowerSoil Total RNA Isolation kit (MoBio, USA) according to the manufacturer’s protocol. Residual DNA was removed with TURBO™DNase (Life Technologies, USA) following the manufacturers recommenda- tions. DNase treated RNA was analyzed using a Bioanalyzer 2100 (Agilent Technologies, USA) and samples with RIN values ≥6 and total RNA concentration ≥100 ng/µl were shipped to BaseClear (Leiden, The Netherlands) for Illumina sequencing (TrueSEQ). BaseClear performed rRNA depletion (RIBO-Zero rRNA Removal Kit (bacteria)) (Epicenter, USA), barcoded cDNA library synthesis (‘dUTP method’71,72) and paired end-sequencing (PE50) and delivered quality trimmed sequence reads. RNA extraction, sequencing and RT-qPCR The remaining part of the DNAse treated RNA samples was used to quantify the genes of interest (GOI) kaiA, kaiB, kaiC, cikA, nifH, psbA and prx of Lyngbya aestuarii PCC8106 (CCY9616) (ccy.nioz.nl) by RT-qPCR with species- speciﬁc primers and TaqMan probes (Table S2, M&M S1). RT-qPCRs were run with technical and biological triplicates. RT-qPCR protocol can be found in the supplementary material (M&M S1). Standard curves were used to estimate GOIs and HKGs concentrations in copies/µl in the original samples. The program BestKeeper73 was used to validate the stability of HKGs. Relative expression values of genes were obtained by normalization against the housekeeping genes rnpA and ppC GOI geomean HKGs ð Þ . An analysis of variance (ANOVA) was performed to assess the GOIs’ regulation. Nevertheless, it is too early to conclude that all microbial mat members possess a clock. Many members of the microbial mat community are still unknown but the prx gene might be a good candidate universal oscillator. The nature of our experimental setup using a natural system under natural conditions, does not allow conclusive proof that light and temperature were the Zeitgebers. However, it is nevertheless likely that these factors entrained the daily gene expression in several mat members as the Cyanobacteria, eukaryotic algae and the Alphaproteobac- teria.20,21 Rhythmic release of organic molecules such as photo- synthate (mainly polysaccharides) during the day and fermentation products such as low molecular weight organic acids (lactate, acetate and ethanol)63 and nitrogen-containing organic compounds during the night64 may serve as additional Zeitgebers. Regulation of gene expression by external stimuli and ﬂuctuating concentrations of substrate is well documented in Bacteria,65,66 while rhythmic synchronization through quorum- sensing, as was shown for Vibrio ﬁscheri,67 could also contribute to gene expression in this complex high diverse and biomass community.68 In conclusion, it was demonstrated that daily rhythmicity of gene expression in a coastal microbial mat surpasses the well- documented circadian clock controlled system of oxygenic phototrophs such as Cyanobacteria and algae. The microbial mat behaves as a well-coordinated consortium of microorganisms with tightly controlled metabolic networks that exhibit the characteristics of a single living entity. The Choirmaster-Choir theory, proposes that light and temperature induced daily rhythmicity in clock possessing phototrophic microorganisms (the choirmaster) is imposed on the microbial mat community as a whole (the choir) through the rhythmic release of metabolites. Published in partnership with Nanyang Technological University DISCUSSION A similar periodicity was found in metatranscriptome studies of marine microbial communities where functional groups of microorganisms other than Cyano- bacteria revealed signiﬁcant differences in gene expression between day and night54 and in daily transcript cycles.22 The observation of community-wide rhythmic gene expression patterns in a coastal microbial mat led us to propose the Choirmaster-Choir theory. This supposes that rhythmic gene expression in microorganisms possessing a circadian clock induces rhythmic gene expression in other microbial mat members. The Choirmaster-Choir theory is derived from three sub-hypotheses: (i) only Cyanobacteria and algae have a fully functional molecular clock and direct their gene expression patterns to other mat members through the rhythmic release of photosynthate and other metabolites; (ii) other microbial mat members have clocks but are only entrained by the rhythmic release of metabolites from phototrophic microorganisms; (iii) other microbial mat members have their own clock that is entrained by a cocktail of Zeitgebers such as light, temperature and the rhythmic release of photosynthate and metabolites by their neighbors. Cyanobacteria In these marine microbial community studies, a large fraction of the cyclic transcripts was derived from Roseobacter, a genus belonging to the Alphaproteobacteria. Our recruitment analysis Daily rhythmicity in coastal microbial mats C Hörnlein et al. 9 Analysis of metatranscriptomes The metatranscriptomic reads were analyzed using two different approaches which resulted in two differently sized datasets. First, RNA reads were clustered into conserved gene transcripts (CGT- data set) of ≥96% identity allowing 2 nucleotide (nt) differences within the 50 nt reads using Vsearch v1.11.174 and transformed to a CGT table depicting the number of reads per CGT per time series. This was done separately for the forward and reverse reads to obtain technical replicates. Both CGT tables served as input for the metaCycle R package that performs statistical tests to identify rhythmic genes with a signiﬁcant periodicity (p ≤0.05) of 20–24 h, reminiscent of a circadian clock. Subsequently, reads were annotated at the DNA level using the Blastn algorhythms to search against the NCBI nt (nucleotide) reference database containing protein coding and structural RNA coding genes and by using BLASTx against nr protein database to retrieve solely protein coding sequences. The R package ‘reutils’ (https://cran.r- project.org/web/packages/reutils/index.html) was used to couple function to taxonomy. Second, reads were uploaded to MG- RAST75 and joined with their pairs where possible. Due to MG- RAST read length restrictions (≥75 bp) only paired reads that merged with a minimum overlap of 8 bp and a maximum difference of 10% were processed (MG-RASTs Fastq-join utility76). Taxonomic annotation was done using the implemented refer- ence databases SILVA SSU for rRNA reads and Genbank (gbk)77 for protein-coding reads with default cut-off values (e-value = 1−e−5, %-identity = 60%). Protein-coding reads were taxonomically ﬁltered and split into ﬁve subsets: Eukarya (Eu), Bacteria (all bacterial reads excluding cyanobacterial reads) (B-C), Cyanobac- teria (C), Proteobacteria (P) and Bacteroidetes (Bs). The last three datasets represent the most abundant bacterial phyla in the metatranscriptomes. Protein annotation of the subsets was performed using the ontology-based SEED subsystem database. Rhythmic genes in this dataset were identiﬁed using metaCycle as described above. In addition, the MG-RAST gbk annotation was used to extract reads annotated as kaiABC, cikA, nifH, psbA and prx for metaCycle analysis. Recruitment analysis 8. Steunou, A.-S. et al. Regulation of nif gene expression and the energetics of N2 ﬁxation over the diel cycle in a hot spring microbial mat. ISME J. 2, 364–378 (2008). The bacterial reference genomes were selected based on their abundance in the MG-RAST dataset, which were found in similar numbers in each sample. Included genomes were Roseobacter denitriﬁcans OCh 114 (NC_008209.1), Congregibacter litoralis KT71 (NZ_CM002299), Allochromatium vinosum DSM 180 (NC_013851), Lyngbya aestuarii PCC8106 (CCY9616) (NZ_AAVU00000000.1) and Coleofasciculus chthonoplastes PCC7420 (CCY9604) (synonym: Microcoleus chthonoplastes; NZ_DS989896.1) (https://ccy.nioz.nl). The annotated genes of these genomes were linked to their matching SEED subsystems using the RAST-server.78 Prior to mapping, the metatranscriptomic reads were depleted of rRNA sequences using sortmeRNA v2.0.79 The rRNA depleted paired-end reads of the six metatranscriptomes were then mapped to the above-mentioned bacterial reference genomes using Bowtie2 v7.1.080 with default parameters in Geneious R8.1.7.81 For down- stream analyses, Geneious was used to normalize mapped reads into transcripts per million (TPM) allowing comparison of gene expression between samples.82 Rhythmic genes in the recruited dataset were identiﬁed using metaCycle as described above. 9. Johnson, C. H. & Egli, M. Metabolic compensation and circadian resilience in prokaryotic cyanobacteria. Annu. Rev. Biochem. 83, 221–247 (2014). 10. Ma, P., Woelﬂe, M. A. & Johnson, C. H. An evolutionary ﬁtness enhancement conferred by the circadian system in cyanobacteria. Chaos, Solitons and Fractals 50, 65–74 (2013). 11. 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Competing interests: The authors declare no competing interests. 30. Wu, G., Anaﬁ, R. C., Hughes, M. E., Kornacker, K. & Hogenesch, J. B. MetaCycle: an 30. Wu, G., Anaﬁ, R. C., Hughes, M. E., Kornacker, K. & Hogenesch, J. B. MetaCycle: an integrated R package to evaluate periodicity in large scale data. Bioinformatics 32, 3351–3353 (2016). integrated R package to evaluate periodicity in large scale data. Bioinformatics 32, 3351–3353 (2016). 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. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. 31. Armitage, D. W., Gallagher, K. L., Youngblut, N. D., Buckley, D. H. & Zinder, S. H. Millimeter-scale patterns of phylogenetic and trait diversity in a salt marsh microbial mat. Front. Microbiol. 3, 293 (2012). Data availability 19. Loza-Correa, M., Gomez-Valero, L. & Buchrieser, C. Circadian clock proteins in prokaryotes: hidden rhythms? Front. Microbiol. 1, 130 (2010). Raw sequencing data is available under the project entitled: ‘Rhythm on the beach’, submitted to MG-RAST project number: mgp9582. https://www.mg-rast.org/linkin.cgi?project=mgp9582 20. Ma, P., Mori, T., Zhao, C., Thiel, T. & Johnson, C. H. Evolution of KaiC-dependent timekeepers: a proto-circadian timing mechanism confers adaptive ﬁtness in the purple bacterium Rhodopseudomonas palustris. PLoS Genet. 12, e1005922 (2016). 21. Min, H., Guo, H. & Xiong, J. Rhythmic gene expression in a purple photosynthetic bacterium, Rhodobacter sphaeroides. FEBS Lett. 579, 808–812 (2005). Recruitment analysis M., Hertel, S., Wiegard, A., Dörrich, A. K. & Wilde, A. 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https://openalex.org/W2985324159	https://europepmc.org/articles/pmc6814890?pdf=render	English	null	Telemonitoring in diabetes: evolution of concepts and technologies, with a focus on results of the more recent studies	Journal of medicine and life	2,019	cc-by	7,788	DOI: 10.25122/jml-2019-0006 DOI: 10.25122/jml-2019-0006 Journal of Medicine and Life Vol. 12, Issue 3, July-September 2019, pp.203–214 Abstract This is a narrative review of telemonitoring (remote monitoring) projects and studies within the field of diabetes, with a focus on results of the more recent studies. Since the beginning of the 1990s, several telemedicine projects and studies focused on type 1 and type 2 diabetes. Over the last 5 years, numerous telemedicine projects based on connected objects and new information and communication technologies (ICT) (elements defining telemedicine 2.0) have emerged or are still under development. Two examples are the DIABETe and Telesage telemonitoring project which perfectly fits within the telemedicine 2.0 framework – the first to include artificial intelligence (AI) with MyPrediTM and DiabeoTM. Mainly, these projects and studies show that telemonitoring diabetic result in: improvements in control of blood glucose (BG) level and significant reduction in HbA1c (e.g., for Telescot et TELESAGE studies); positive impact on co-morbidities (arterial hypertension, weight, dyslipidemia) (e.g., for Telescot and DIABETe studies); better patient’s quality of life (e.g., for DIABETe study); positive impact on appropriation of the disease by patients and/or greater adherence to therapeutic and hygiene- dietary measures (e.g., The Utah Remote Monitoring Project); and at least, good receptiveness by patients and their empowerment. To date, the magnitude of its effects remains debatable, especially with the variation in patients’ characteristics (e.g., background, ability for self-management, medical condition), samples selection and approach for the treatment of control groups. All of the recent studies have been classified as “Moderate” to “High”. Keywords: artificial intelligence, chronic disease, diabetes, information and communication technology, Internet, telemedicine, telemonitoring, Web home BG monitoring, non-adherence or non-compliance with medications or lifestyle changes (nutrition and sport), sub-optimal patient education about the disease, and limited access to health professionals [2]. Telemonitoring in diabetes: evolution of concepts and technologies, with a focus on results of the more recent studies Emmanuel Andrès1,2, Laurent Meyer3, Abrar-Ahmad Zulfiqar2,4, Mohamed Hajjam5, Samy Talha2,6, Thibault Bahougne3, Sylvie Ervé7, Jawad Hajjam7, Jean Doucet4, Nathalie Jeandidier3, Amir Hajjam El Hassani8 1. Service de Médecine Interne, Diabète et Maladies Métaboliques de la Clinique Médicale B, Hôpitaux Universitaires de Strasbourg, 1, porte de l’Hôpital, 67091 Strasbourg cedex France 2. Equipe de recherche EA 3072 «Mitochondrie, Stress oxydant et Protection musculaire», Faculté de Médecine de Strasbourg, Université de Strasbourg (Unistra), Strasbourg, France 3. Service d’Endocrinologie et de Diabétologie de la Clinique Médicale B, Hôpitaux Universitaires de Strasbourg, Strasbourg, France 4. Service de Médecine Interne, Gériatrie et Thérapeutique, CHU de Rouen, France 5. Predimed Technology, Strasbourg, France 6. Service de Physiologie et d’Explorations Fonctionnelles, Hôpitaux Universitaires de Strasbourg, Strasbourg, France 7. Centre d’expertise des Technologies de l’Information et de la Communication pour l’autonomie (CENTICH) et Mutualité Française Anjou-Mayenne (MFAM), Angers, France 8. Equipe de recherche EA 4662 «Nanomédecine, Imagerie, Thérapeutiques», Université de Technologie de Belfort-Montbéliard (UTBM), Belfort-Montbéliard, France Corresponding Author: Emmanuel Andrès Service de Médecine Interne, Diabète et Maladies Métaboliques de la Clinique Médicale B, Hôpitaux Universitaires de Strasbourg, 1, porte de l’Hôpital, 67091 Strasbourg cedex France, E-mail: emmanuel.andres@chru-strasbourg.fr Received: January 29th, 2019 – Accepted: March 8th, 2019 Received: January 29th, 2019 – Accepted: March 8th, 2019 Search Strategy A literature search has been performed on the PubMed database of the US National Library of Medicine (https:// www.ncbi.nlm.nih.gov/pubmed) and on Scholar Google (https://scholar.google.fr/). We searched for articles published between January 1990 and December 2018, using the following key words or associations: “diabetes mellitus”, “telemedicine” and “telemedicine in diabetes mellitus”; restrictions included: language (“English” or “French”); and type of publication (“Clinical trials”, “Review articles” and “Guidelines”). Textbooks on telemedicine and information gleaned from international meetings were also used. Introduction Intensive glucose control has been shown to delay or prevent the development of micro- and macro-vascular complications related to diabetes. However, it is estimated that 43.2–55.6% of adults with type 2 diabetes do not meet the reference target for glycemic control (hemoglobin A1c [HbA1c] <7.0%) [1]. Factors that may contribute to sub- optimal blood glucose (BG) control include inadequate In this context, telemedicine may be an effective approach to solve problems of education, compliance, and monitoring and provider access [3]. BG control could be safely improved by changing the drugs on home BG readings and transmitting them in near-real time to 203 Journal of Medicine and Life Vol. 12, Issue 3, July-September 2019 true effect is probably markedly different from the estimated effect”; “Low = The true effect might be markedly different from the estimated effect”; “Moderate = The authors believe that the true effect is probably close to the estimated effect”; “High = The authors have a lot of confidence that the true effect is similar to the estimated effect”. providers. In this setting, telemedicine may also be an effective solution to monitor the complications of the diabetes, especially macro-vascular complications (e.g., myocardial infarction, heart failure) and co-morbidities (e.g., arterial hypertension). In this article, we review with a pragmatic mind and a clinical vision in the field of remote monitoring (telemonitoring) of diabetic patients. We have reviewed 201 references, which yielded 85 potentially relevant papers. After selection, only 29 papers have been included in our narrative review (defined as a comprehensive, critical and objective analysis of the current knowledge on a topic) and analyzed. Only published telemedicine trials or studies including a clinical evaluation, potentially useful to the clinician in everyday practice have been included in this review. It is to note that the present narrative review is limited by its focus on non-invasive telemonitoring in diabetic patients. Telemedicine Studies from 2010 to 2015 For the majority of patients, they conducted on: Since the 2010–2015, more numerous mature telemedicine projects and studies have been developed in the setting of diabetes management, especially in the setting of telemonitoring [29–32]. These projects and studies have a main objective to evaluate the use of technology to implement medical and cost-effective healthcare management on a large scale for diabetes management.i – Type 1 and type 2 diabetic patients (e.g., children and young people [n=3] or elderly patients [n=2]);i – Patients with intensified therapy (at least two oral anti-diabetic agents + insulin therapy) (n=2) or under insulin pump therapy (n=1); – Patients with complicated or complex diabetes (e.g., co-morbidities and cardiovascular complications) (n=2). All of these studies were GRADE classified as “Moderate” [29–31] except the Telescot Diabetes Pragmatic Multicenter Randomized Controlled Trial classified as “High” [32]. These studies have involved telemonitoring through the upload and direct transmission of BG data by diabetic patients to providers via cell phone, telephone land line, or Internet-based program [4–27]. Compared to the aforementioned projects, most of these have incorporated several new tools or processes for a better management of the diabetic patient, they are as follows: The results of these telemedicine projects differed from study to study, with fairly inconclusive results, particularly regarding the statistical significance of the results [3, 28]. In view of their objectives, these studies did not make it possible to conclude on the usefulness of telemedicine in terms of diabetes equilibration and management. One of the explanations for these results is the inclusion of relatively well-balanced diabetic patients. Nevertheless, some studies were particularly promising. This is the case of the DiaTel study [26, 27], GRADE classified as “Moderate”. – Tools or processes for medical education, particularly for patient ownership of the disease, better food hygiene, and increased physical activity; – Tools for therapeutic and hygiene observance; – Tool to remote co-morbidities (e.g., arterial hypertension, obesity, dyslipidemia); – Tools for interaction between the patient and healthcare professionals like telephone support centers, tablets, and Internet-sites, smartphones (Figure 1) [29–32]. The DiaTel study compared the short-term efficacy of home telemonitoring coupled with active medication management by a nurse practitioner, with a monthly care coordination telephone call on glycemic control, in veterans with type 2 diabetes [26]. The included patients were taking oral hypoglycemic agents and/or insulin for ≥1 year and had HbA1c ≥7.5%). Telemedicine Studies from 1990 to 2010 Since the early 1990s to the end of 2010, more than 20 telemedicine projects and studies have been developed in the field of diabetes and its management [4–27]. Practically all of them have investigated “structured-telephone support” (defined as a remote management that can be provided through structured telephone contact between patients and healthcare providers – with or without home visits and reporting of symptoms and/or physiological data) or “telemonitoring” (defined as the use of information technology to monitor patients at a distance). These studies have been designed only to monitor BG levels (Figure 1) [4–27]. The first were more like “proof-of-concept Our main objective is to provide practical information to clinicians on the benefits of telemonitoring, documented from the current medical literature data. In this context, the selected studies have been classified using the GRADE tool (Grading of Recommendations, Assessment, Development and Evaluations) to indicate the level of evidence (https:// bestpractice.bmj.com/info/us/toolkit/learn-ebm/what-is- grade/). The GRADE tool proposes an evidence classification ranging from very low to high, as follows: “Very low = The Figure 1: Telemonitoring in diabetes: evolution of concepts and technologies, with a focus on results of the more recent studies. Figure 1: Telemonitoring in diabetes: evolution of concepts and technologies, with a focus on results of the more recent studies. 204 Journal of Medicine and Life Vol. 12, Issue 3, July-September 2019 months (1.7% vs. 0.8%; p<0.001 for each), improvement occurring by 3 months. studies” (defined as a demonstration in principle with the aim of verifying that some concept or theory has practical potential). All of these studies were GRADE classified as “Very low” or “Low”. Telemedicine Studies from 2010 to 2015 The device was programmed to sound an alarm at a pre-specified patient-referred time to prompt the patient to initiate a telemonitoring session g – Patient received a series of education messages, focused on teaching patients about their diseases (diabetes, arterial hypertension) and associated co-morbidities Second arm: – Remote monitoring device: blood glucose level, blood pressure, heart rate and weight and telemonitoring with an interactive voice response (IVR) system ( ) y – Patient received a call from the telemonitoring IVR service at a pre-specified time. Medical providers were contacted either via a note in the electronic medical record (or immediately if there was a concern, in person or by telephone) if there was an out-of-range value (decided by individual providers or clinics as a value that was high or low) Randomized Trial on Home Telemonitoring for the Management of Metabolic and Cardiovascular Risk in Patients with type 2 Diabetes (n=302) [30] Randomized, parallel-group, open-label, multicenter study Type 2 diabetic patients in general medicine – Remote monitoring device: blood glucose level, blood pressure, heart rate and weight. The telemonitoring system is associated with remote educational support and feedback to the general practitioner Study assessed the utility and cost- effectiveness of an automated Diabetes Remote Monitoring and Management System (DMRS) (n=98) [31] Randomized, controlled study Patients with uncontrolled diabetes on insulin – DRMS use text messages or phone calls to remind patients to test their blood glucose and to report results via an automated system. The DRMS made adjustments to insulin dose(s) based on validated algorithms Telescot Diabetes Pragmatic Multicenter Randomized Controlled Trial (n=321) [32] Randomized, parallel, investigator- blind controlled trial Patients with relatively well-controlled type 2 diabetes, with an HbA1c > 7.46% – Telemonitoring intervention involved self-measurement and transmission to a secure website (weekly twice, morning and evening blood glucose level) – Remote monitoring device: blood glucose level, blood pressure, heart rate and weight. The telemonitoring system is associated with remote educational support and feedback to the general practitioner – DRMS use text messages or phone calls to remind patients to test their blood glucose and to report results via an automated system. The DRMS made adjustments to insulin dose(s) based on validated algorithms ambulatory diastolic BP was 2.17 mmHg lower (95% CI 0.62–3.72, p=0.006) among people in the intervention group when compared with usual care after adjustment. Telemedicine Studies from 2010 to 2015 At enrollment, the patients were randomly assigned to either: active care management (AMC) with home telemonitoring (HT) (ACM + HT group, n=73); and a monthly care coordination telephone call (CC group, n=77) [27]. Both groups received monthly calls for diabetes education and self-management review. ACM + HT group participants transmitted BG level, blood pressure (BP), and weight to a nurse practitioner; the nurse practitioner adjusted medications for glucose, BP, and lipid control based on established American Diabetes Association targets. Baseline characteristics of the patients in the DiaTel study were similar in both groups, with mean HbA1c: 9.4% in the CC group vs. 9.6% in ACM + HT group [26, 27]. Compared with the CC group, the ACM + HT group demonstrated significantly larger decreases in HbA1c (principal criterion) at 3 months (1.7% vs. 0.7%) and 6 The characteristics and the results of the main telemonitoring studies conducted in diabetic patients during this period (2010–2015) are reported in Tables 1 and 2 [29–32]. Analysis of these tables shows that at least three of the studies are positive for improving glycemic control (e.g., BG levels and/or HbA1c) and improving co-morbidities (e.g., arterial hypertension). In this setting the Telescot Diabetes Pragmatic Multicenter Randomized Controlled Trial is the most clinically convincing and methodologically sound study [32]. The Telescot study is a randomized, parallel, investigator-blind controlled trial with centralized randomization in family practices in four regions of the United Kingdom (Table 1) [32]. This study included 321 patients with relatively well-controlled type 2 diabetes, with an HbA1c >7.46%. In Telescot Diabetes, 160 people were randomized to the intervention group and 161 to the usual care group. The supported telemonitoring intervention involved self-measurement and transmission to a secure website – twice weekly morning and evening glucose – for 205 Journal of Medicine and Life Vol. 12, Issue 3, July-September 2019 1: Characteristics of the telemonitoring studies conducted in the field of diabetes during the period from 2010 to 2015 Type of telemonitoring First arm: – Remote monitoring device: blood glucose level, blood pressure, heart rate and weight. The device was programmed to sound an alarm at a pre-specified patient-referred time to prompt the patient to initiate a telemonitoring session – Remote monitoring device: blood glucose level, blood pressure, heart rate and weight. Telemedicine Studies from 2010 to 2015 18.5% (p=0.07)i – No difference had been registered for body weight, BP, and lipid profile Secondary criteria:i – For quality of life (evaluated with the 36-item Short Form health survey), significant differences in favor of the telemonitoring group, as for physical functioning (p=0.01) and mental health (p=0.005) – On an economic level, a lower number of specialist visits was reported in the telemedicine group: incidence rate ratio of 0.72 (95% confidence interval, 0.51–1.01; p=0.06) – No significant difference for mean HbA1c between the DRMS and control groups at 3 months: 7.60% vs. 8.10% and at 6 months: 8.10% vs. 7.90% (p=ns) Secondary criteria: y – Changes from baseline to 6 months have been not statistically significant for self-reported medication adherence – Changes of diabetes-specific quality of life have been not significant registered, except for the Daily Quality of Life-Social/Vocational Concerns subscale score (p=0.04) p – The Mean (SD) HbA1c at follow-up was 7.92% in the intervention group vs. 8.36% in the usual care group]. For primary analysis, adjusted mean HbA1c was 0.51% lower (95% CI 0.22% to 0.81%, (principal criterion) (p=0.0007) – Adjusted mean ambulatory systolic BP has been 3.06 mmHg lower (95% CI 0.56–5.56 mmHg, p=0.017) and mean ambulatory diastolic BP has been 2.17 mmHg lower (95% CI 0.62–3.72, p=0.006) among people in the intervention group when compared with usual care after adjustment g p p g p p j – No significant differences were identified between groups in terms of: weight, treatment pattern, adherence to medication or quality of life y – The number of telephone calls was greater between nurses and patients in the intervention compared with control group: rate ratio of 7.50 (95% CI 4.45–12.65, p<0.0001) but no other significant differences between groups in use of health services were identified between groups Telemedicine Studies from 2010 to 2015 No significant differences were identified between groups in weight, treatment pattern, adherence to medication, or quality of life in secondary analyses. During the study, the number of telephone calls was greater between nurses and patients in the intervention compared with the control group: rate ratio of 7.50 (95% CI 4.45–12.65, p<0.0001) but no other significant differences between groups (in use of health services) were identified between groups. review by family practice clinicians who were not blinded to allocation group. The control group received usual care, with at least annual review and more frequent reviews for people with poor glycemic or BP control. HbA1c assessed at 9th month was the primary outcome. The mean (SD) HbA1c at follow-up was 7.92% in the intervention group vs. 8.36% in the usual care group (Table 2). For primary analysis, adjusted mean HbA1c was 0.51% lower (95% CI 0.22%–0.81%, p=0.0007). For secondary analyses, adjusted mean ambulatory systolic BP was 3.06 mmHg lower (95% CI 0.56–5.56 mmHg, p=0.017) and mean 206 Journal of Medicine and Life Vol. 12, Issue 3, July-September 2019 Table 2: Results of the telemonitoring studies conducted in the field of diabetes during the period from 2010 to 2015 p – Mean HbA1c had decreased from 9.73% at baseline to 7.81% at the end of the program (p<0.0001) 1c p g (p ) – Systolic blood pressure (BP) had decreased from 130.7 mmHg at baseline to 122.9 mmHg at the end (p=0.0001) Secondary criteria: y – Low-density lipoprotein content had decreased from 103.9 mg/dl at baseline to 93.7 mg/dl at the end (p=0.0263)i – Knowledge of diabetes and arterial hypertension have increased significantly (p<0.001 for both) – Patient engagement and medication adherence also have improved, but not significantly g yp gi y (p ) – Patient engagement and medication adherence also have improved, but not significantly – Per questionnaires at study end, patients felt the telemonitoring program had been useful Principal criteria: – Mean HbA1c difference of 0.33±0.1 (p=0.001) have been observed between the telemonitoring compared and the control group. The proportion of patients reaching the target of HbA1c (HbA1c <7.0%) had been higher in the telemonitoring group than in the control group after 6 months: 33.0% vs. 18.7% (p=0.009) and 12 months: 28.1% vs. Telemedicine Studies from 2015 to the Present they will utilize new Information and Communication Technologies (ICT) (defined as the infrastructure and components that enable modern computing) and the Web 2.0 technologies (defined as a renewal or evolution of these older technologies or of the Internet itself), later being based on old technologies such as HTML) [37]. Over the last 5 years, “new generation” telemedicine projects and studies have emerged in the setting of type 1 and type 2 diabetes [3, 33–36]. They support automatic transmission and remote interpretation of patients’ data for follow-up and preventive interventions (Figure 1). These new generation telemedicine projects are often known as “telemedicine 2.0” projects (also called “e-Health 2.0”), Most studies have been constructed on various connected tools (Bluetooth and/or Wi-Fi) for monitoring type 1 and type 2 diabetes and its co-morbidities, such as glucose meters, BP monitors, heart rate monitors, weighing 207 Journal of Medicine and Life Vol. 12, Issue 3, July-September 2019 scales, and pulse oximeters [33–36]. Several projects also include continuous glycemic monitoring solution connected tablets and smartphones and often a video-call [3]. Several of these telemedicine projects use machine learning, also called artificial intelligence (AI), in order to be able to: whether the introduction of a health technology-supported self-management program involving telemonitoring and health counseling had beneficial effects on HbA1c, other clinical variables (weight, body mass index, BP, blood lipid profile), and Health-Related Quality of Life (HRQoL), as measured using the Short Form Health Survey 36 (SF-36) in patients with type 2 diabetes (Figure 2) [33]. – Adjust the BG level to the patient’s activity (software DiabeoTM) [34, 35]; This was a pragmatic randomized controlled trial of patients with type 2 diabetes. Both the control (n=79) and intervention groups (n=87) received usual care [33]. The intervention group also participated in additional health promotion activities with the use of the Prescribed Healthcare Web application for self-monitoring of BG and BP. About every second month or when needed, the general practitioner or the DM nurse reviewed the results and the healthcare activity plan. Analysis of the data showed that there were no significant differences between the groups in the primary outcome HbA1c level (p=0.33), and in the secondary outcome HRQoL as measured using SF-36. A total of 80% of the patients in the intervention group at the baseline, and 98% of the responders after 19-month intervention were familiar with using a personal computer (p=0.001). Telemedicine Studies from 2015 to the Present After 19 months, no responders reported significantly poorer mental health in social functioning and emotional role subscales on the SF-36 (p=0.03, and p=0.01, respectively). – Predict patient risks of diabetes decompensation [36]. In the later situation, the cloud-based software aggregates, cleans, and analyzes patient data to allow for identifying patterns that may indicate potential risks and provide predictive insights on healthcare outcomes, as the software MyPrediTM [36, 38]. In fact, several informatics solutions or tools have been developed and used in chronic diseases monitoring (as diabetes), such as Artificial Neural Networks (ANN) algorithms, data mining software, ontology [38]. Besides these tools, it must be emphasized that diabetes telemonitoring may use, as for CHF telemonitoring, implantable invasive devices that send either sporadically or continuous data to the receiving physician (automatic telemonitoring) (outside the scoop of this paper) [3, 28]. In management of diabetes, implantable telemonitoring devices for multi-parameters including mainly BG-insulin levels monitoring have recently proven to be an effective approach. TELESAGE Study TELESAGE (“Suivi A Grande Echelle d’une population de diabétiques de type 1 et de type 2 sous schéma insulinique basal bolus par la TELEmédecine”) is a 6-month open-label parallel-group, multicenter study, including adult diabetic patients (n=180), with type 1 diabetes (>1 year), on a basal- bolus insulin regimen (>6 months), with an HbA1c ≥8% [34, 35]. This study will compare a control group (group 1 [G1]: usual follow-up) with two DiabeoTM telemedicine All of the studies conducted from 2015 to the present were GRADE classified as “Moderate” [33, 39, 40], except the TELESAGE study classified as “High” [34, 35]. Telemonitoring and health counseling for self- management support of patients with type 2 diabetes The objective of the Telemonitoring and Health Counseling for Self-Management Support study was to investigate Figure 2: Telemonitoring devices and information flow during the field trial (adapted from [33]). Figure 2: Telemonitoring devices and information flow during the field trial (adapted from [33]). 208 Journal of Medicine and Life Vol. 12, Issue 3, July-September 2019 Journal of Medicine and Life Vol. 12, Issue 3, July-September 2019 systems: (1) physician-assisted telemedicine (G2), and (2) nurse-assisted telemonitoring and teleconsultations by a diabetologist’s task delegation (G3) (Figure 3). Figure 3: TELESAGE process for diabetic patients assigned to arm 3: (i) Self-measured plasma glucose levels before and after meals (6 measurements) + 1 optional in the night; (ii) carbohy drate counts; and (iii) planned physical activity. HCP: healthcare practitioner (adapted from [34]). At 6-month, the mean HbA1c levels were significantly different between the three arms of the TELESAGE study: 8.41±1.04% in G3 vs. 8.63±1.07% in G2 vs. 9.10±1.16% in G1 (p=0.0019 for G1–G3 comparison) (Figure 4) [34, 35]. The DiabeoTM system gave a 0.91% (0.60–1.21) improvement in HbA1c over controls and a 0.67% (0.35–0.99) reduction when used without teleconsultation. There was no difference in the frequency of hypoglycemic episodes or in medical time spent for hospital or telephone consultations. However, patients in G1 and G2 spent nearly 5 h more than G3 patients attending hospital visits. DIABETe Project Figure 3: TELESAGE process for diabetic patients assigned to arm 3: (i) Self-measured plasma glucose levels before and after meals (6 measurements) + 1 optional in the night; (ii) carbohy drate counts; and (iii) planned physical activity. HCP: healthcare practitioner (adapted from [34]). The DIABETe project has been developed to optimize home monitoring of diabetic patients via a telemonitoring 2.0 platform, situations with a risk of decompensation of diabetes and its cardiovascular complications (e.g., myocardial infarction or chronic heart failure [CHF]), the latter ultimately leading to hospitalization [3, 39]. The AI of the DIABETe platform automatically generates indicators Figure 4: Efficacy of the software DiabeoTM, licensed by Sanofi Laboratory. A: HbA1c values (means±SE), from 3 months before base line to month 6. p=0.0103, p=0.0019 compared with control group. B: Change in HbA1c values (means±SE) from baseline to month 6 (adapted from [34, 35]). Figure 4: Efficacy of the software DiabeoTM, licensed by Sanofi Laboratory. A: HbA1c values (means±SE), from 3 months before base line to month 6. p=0.0103, **p=0.0019 compared with control group. B: Change in HbA1c values (means±SE) from baseline to month 6 (adapted from [34, 35]). 209 Journal of Medicine and Life Vol. 12, Issue 3, July-September 2019 of “health status” deterioration, i.e., “warning alerts” for any chronic disease worsening, particularly diabetes. The platform comprises connected non-intrusive medical sensors (Figure 5), a touchscreen tablet connected by Wi-Fi, and a router or 3G/4G, rendering it possible to interact with the patient and provide education on treatment, diet, and lifestyle. The system involves a server that hosts the patient’s data and a secure Internet portal to which the patient can be connected to hospital- and non-hospital-based healthcare professionals. DIABETe is based on a smart system comprising an inference engine and a medical ontology for personalized synchronous or asynchronous analysis of data specific to each patient and, if necessary, the sending of an AI-generated alert (MyPrediTM). that MyPrediTM detected any worsening of the “patient’s health”, with a sensitivity, specificity, as well as positive and negative predictive values of: 100%, 30%, 89% and 100%, respectively. In this experimentation, both the healthcare professionals and patients, even the frailest, used the E-care system without difficulty until the end of the study. Integration of the problems of elderly subjects and co-morbidities associated with diabetes The challenge for “tomorrow’s” telemedicine is to develop new telemedicine projects and solutions, including the resolution of several medical problems and difficulties, such as [3]:i – The specificities (no appetite for new technologies and uses) and problems (e.g., falls, malnutrition, mild cognitive impairment, etc.) of elderly subjects, who are the main subjects affected by chronic diseases; The telemonitoring platform used in DIABETe was first validated in a monocentric study conducted in the Strasbourg University Hospital, carried out as part of the E-Care project, primarily focused on the problem of CHF [39, 40]. One hundred and seventy-five patients (mean age of 72 years) were included into the E-care project, 30% of the patients suffered from type 2 diabetes. During this period, the telemonitoring platform was used on a daily basis by patients and healthcare professionals according to a defined protocol of use which is specific to each patient. During the study, 1500 measurements were taken to generate 700 alerts in 68 patients. One hundred and seven subjects (61.1%) had no alerts upon follow-up. Analysis of the warning alerts in the 68 other patients showed – The co-existence of several chronic pathologies (e.g., diabetes, CHF, COPD, etc.) and co-morbidities (e.g., arterial hypertension, renal failure, etc.) in the same individual, while providing comprehensive and “global” care for the individual in all medical and societal dimensions; – The multiplicity of care structures and medical organizations (e.g., with or without human resources, telemedical centers, etc.); 210 Figure 5: Telemedicine project: DIABETe. A: DIABETe is based on a smart system comprising an inference engine and a medical ontology for personalized synchronous or asynchronous analysis of data specific to each patient and, if necessary, the sending of an artificial intelligence-generated alert (MyPrediTM. B: The platform comprises connected non-intrusive medical sensors, a touchscreen tablet connected by Wi-Fi, and a router or 3G/4G, rendering it possible to interact with the patient and provide education on treatment, diet, and lifestyle. C: The system involves a server that hosts the patient’s data and a secure internet portal to which the patient can be connected to hospital- and non-hospital-based healthcare professionals. Figure 5: Telemedicine project: DIABETe. A: DIABETe is based on a smart system comprising an inference engine and a medical ontology for personalized synchronous or asynchronous analysis of data specific to each patient and, if necessary, the sending of an artificial intelligence-generated alert (MyPrediTM. Results of Systemic Reviews and Meta-Analysis To our knowledge, several recent systemic reviews and meta-analysis have been published in the last few months [42–45]. Main results of these works are in accordance with the results of our review. In the work from Wu et al. [42], 19 randomized controlled trials were selected (n = 6294). Telehealth was more effective than usual care in controlling the glycemic index in diabetes patients (weighted mean difference: –0.22%; 95% CI, -0.28–-0.15; p < 0.001). This intervention showed promise in reducing systolic blood pressure levels (p < 0.001) and diastolic blood pressure levels (p <0 .001), while no benefits were observed in the control of body mass index (BMI) (p = 0.79). For total cholesterol and quality of life, telehealth was similar or superior to usual care. In this setting, additional personnel and specific protocols are necessary, which must be specific for each chronic disease and targeted for each patient, while allowing the possibility for each patient to exhibit more than one chronic disease. Most of these protocols must still be funded by means of existing resources or external grants. These diseases share a number of commonalities with diabetes in terms of epidemiology and natural history. Along with diabetes, CHF and COPD are among the most common diseases in developed countries, and thus represent a major public health concern for society [1, 2]. They are accompanied by frequent hospital admissions and re-admissions for well-known causes. These causal factors are detectable, enabling professionals to act ahead of time, as with diabetes and its co-morbidities, thereby avoiding disease progression. Developing warning alerts for these chronic diseases should enhance the existing system. In the review of Hu et al. (14 trials, n=1,324) [44], the use of telemedicine was found to improve HbA1c and reduce the risk of hypoglycemia in diabetic patients, compared to usual care, but without significant difference in BMI. Compared to usual care, telemedicine was found to reduce the odds of hypoglycemia (–0.42; 95% CI = 0.29– 0.59; I2 = 32%; p<0.00001). Hu et al. found that the clinical relevance declined in HbA1c level compared to control group (mean difference=–0.28; 95% CI = -0.45–-0.12; p=0.0005), but the telemedicine had no effect on BMI (mean difference = –0.27; 95% CI = –0.86–0.31; p=0.35). These points have been addressed by the Whole System Demonstrator cluster randomized trial [41]. Integration of the problems of elderly subjects and co-morbidities associated with diabetes B: The platform comprises connected non-intrusive medical sensors, a touchscreen tablet connected by Wi-Fi, and a router or 3G/4G, rendering it possible to interact with the patient and provide education on treatment, diet, and lifestyle. C: The system involves a server that hosts the patient’s data and a secure internet portal to which the patient can be connected to hospital- and non-hospital-based healthcare professionals. Figure 5: Telemedicine project: DIABETe. A: DIABETe is based on a smart system comprising an inference engine and a medical ontology for personalized synchronous or asynchronous analysis of data specific to each patient and, if necessary, the sending of an artificial intelligence-generated alert (MyPrediTM. B: The platform comprises connected non-intrusive medical sensors, a touchscreen tablet connected by Wi-Fi, and a router or 3G/4G, rendering it possible to interact with the patient and provide education on treatment, diet, and lifestyle. C: The system involves a server that hosts the patient’s data and a secure internet portal to which the patient can be connected to hospital- and non-hospital-based healthcare professionals. 210 Journal of Medicine and Life Vol. 12, Issue 3, July-September 2019 – The significant logistical barriers to implementing tele-health (Many existing health systems are not designed for these technologies to be integrated within existing information systems.). expenses) [28, 36]. The growth of value-based payment models may, however, provide incentives to implement telehealth as a strategy for providing high-quality, cost- effective and coordinated care [36]. At country levels, differences in medical practice laws, restrictions on how telehealth can be delivered, and which patients are to receive these services limits the telemedicine applicability as well. In the chronic disease setting, new remote sensors and tailored questionnaires are presently being integrated into the telemedicine platform (e.g., E-care or DIABETEs solutions for our team), including remote actimeters and electronic spirometers, along with new knowledge in the form of ontologies in order to enhance the telemedicine platform and broaden its utility to other chronic diseases like COPD [44, 47].i Results of Systemic Reviews and Meta-Analysis In this study, 3230 people with diabetes, COPD, or CHF were recruited from practices by 179 general practices in three areas in England. The patients were divided into 2 groups: usual care or telehealth with remote exchange of data between patients and healthcare professionals as part of patients’ diagnosis and management. In this trial, telehealth has been associated with lower mortality and emergency admission rates. Compared with controls, the intervention group had a lower admission proportion within 12 month follow-up (odds ratio 0.82, 95% confidence interval 0.70–0.97, p=0.017). Mortality at 12 months was also lower for intervention patients than for controls: 4.6% vs. 8.3% (odds ratio 0.54, 0.39–0.75, p<0.001). In the review of Lee et al. [45], the use of telemedicine for retinal screening was beneficial and cost-effective for diabetes management with an incremental cost- effectiveness ratio between $113.48/quality-adjusted life year (QALY) and $3,328.46/QALY (adjusted to 2017 inflation rate). Similarly, the use of telemonitoring and telephone reminders was cost-effective in diabetes management. Future research must also focus on the accessibility and practicality of telemedicine interventions. Reimbursement also remains a major concern and a barrier (“glass ceiling”), because much of the care delivered by telehealth is not covered by traditional fee-for-service payment models (e.g., in France, where all HF patients benefit from an integrated processing of healthcare Conclusions This narrative review in the field of telemonitoring with a focus on the more recent studies supports its efficacy 211 Journal of Medicine and Life Vol. 12, Issue 3, July-September 2019 in diabetic patients. In this setting, close management of type 1 and type 2 diabetic patients through telemonitoring showed: improvements in control of BG level and significant reduction in HbA1c (e.g., for Telescot et TELESAGE studies [32, 34, 36]); positive impact on co-morbidities (arterial hypertension, weight, dyslipidemia) (e.g., for Telescot and DIABETe studies [32, 39, 40]); better patient’s quality of life (e.g., for DIABETe study) [39, 40]); positive impact on appropriation of the disease by patients and/or greater adherence to therapeutic and hygiene-dietary measures (e.g., The Utah Remote Monitoring Project [29]); and at last, good receptiveness by patients and patient empowerment (Table 3). To date, the magnitude of its effects remains debatable, especially with the variation in patients’ characteristics (e.g., background, ability for self- management, medical condition), samples selection and approach for treatment of control groups . Table 3: Results of the main recent studies on telemonitoring diabetic patients. The Utah Remote Monitoring Project [29] Randomized Trial on Home Telemonitoring for the Management of Metabolic and Cardiovascular Risk in Patients with type 2 Diabetes [30] Study assessed the utility and cost-effectiveness of an automated Diabetes Remote Monitoring and Management System (DMRS) [31] Telescot Diabetes Pragmatic Multicenter Randomized Controlled Trial [32] Telemonitoring and Health Counseling for Self-Management Support study [33] TELESAGE [34, 35] DIABETe [39, 40] Impact on blood-glucose and/or HbA1c levels + + - + - + • Impact on arterial hypertension, weight, dyslipidemia and/or other comorbidities + • - + - • + Impact on quality of life + + - - • + Impact on appropriation of the disease by patients and/or greater adherence to therapeutic and hygiene-dietary measures + • - - • • • Grade classified Moderate Moderate Moderate High Moderate High High “+”: positive impact; “-”: no positive impact; “•”: not studied. Moderate All of these recent studies have been GRADE classified as “Moderate” (the authors believe that the true effect is probably close to the estimated effect) to “High” (The authors have a lot of confidence that the true effect is similar to the estimated effect). Consent and Ethical approval Not applicable. Conclusions To date, relatively few projects and trials in diabetic patients have been run within the “telemedicine 2.0” setting, particularly using AI, ICT and the Web 2.0, as for the studies TELESAGE and DIABETe [34, 35, 39, 40]. Moderate Further investigations are needed on efficacy and cost-effectiveness over longer periods of time, and larger samples of diabetic patients. Funding Grants from the Fondation de l’Avenir, the Agence Régionale de Santé du Grand-Est (ARS) and the Agence Nationale de la Recherche (ANR). References 19. Shea S, Weinstock RS, Starren J, Teresi J, Palmas W, Field L, et al. 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Van De Belt TH, Engelen L, Berben S, Schoonhoven L. Definition of health 2.0 and medicine 2.0: a systematic review. J Med Internet Res. 2010;12:e18. 28. Puricel SG, Ruiz J. Le diabète et l’ère de la télémédecine. Rev Med Suisse. 2014;10:1246–8. 38. Benyahia A, Hajjam A, Andrès E, Hajjam M, Hilaire V. Including other system in E-Care telemonitoring platform. Stud Health Technol Inform. 2013;190:115–7. 29. Shane-McWhorter, Lenert L, Petersen M, et al. The Utah Remote Monitoring Project: improving health care one patient at a time. Diabetes Technol Ther. 2014;16:653–60. 39. Andrès E, Talha S, Hajjam M, Hajjam J, Ervé S, Hajjam A. References Hu Y, Wen X, Wang F, Yang D, Liu S, Li P1, et al. Effect of telemedicine intervention on hypoglycaemia in diabetes patients: a systematic review and meta-analysis of randomised controlled trials. J Telemed Telecare. 2018;1:1357633X18776823. doi: 10.1177/1357633X18776823. [Epub ahead of print] 36. 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W4387772168.txt	https://periodicorease.pro.br/rease/article/download/11263/5049	pt		SATISFAÇÃO DE USUARIOS COM RELAÇÃO ÀS CONDIÇÕES HIGIÊNICO–SANITÁRIAS DA ALIMENTAÇÃO DO MERCADO PÚBLICO ISAEL ALMEIDA EM FLORIANO-PI	Revista Ibero-Americana de Humanidades, Ciências e Educação	2,023	cc-by	4,995	Revista Ibero- Americana de Humanidades, Ciências e Educação- REASE doi.org/10.51891/rease.v9i9.11263 SATISFAÇÃO DE USUARIOS COM RELAÇÃO ÀS CONDIÇÕES HIGIÊNICO– SANITÁRIAS DA ALIMENTAÇÃO DO MERCADO PÚBLICO ISAEL ALMEIDA EM FLORIANO-PI Marisa Elanne Damasceno de França1 Kelly Regina Alves de Oliveira2 Laura Cibelle Damasceno Duarte3 Elda de Sousa Pimentel4 Raisa de Sá Veloso5 Jocilene Mesquita Saraiva6 Kamilla Magalhães Torres Lima7 Luana Clementino Rodrigues8 RESUMO: As doenças transmitidas por água e alimentos são ainda, um grave problema de saúde pública no mundo contemporâneo, mesmo com a evolução tecnológica das últimas décadas quanto às técnicas de conservação e higiene dos alimentos, aonde os alimentos são reconhecidos como o principal vetor das enfermidades entéricas agudas. As condições higiênico-sanitárias de alimentos são de grande importância em qualquer ambiente, visto que diversos são os perigos tanto biológicos, como físicos e químicos que podem originar doenças transmissíveis por alimentos. Diversas enfermidades podem ser evitadas quando a manipulação de alimentos está dentro das normas técnicas de higiene nas etapas de pré-preparo e preparo. De acordo com os princípios de higiene ambiental, pessoal e operacional deve-se garantir a ausência de contaminações de qualquer origem. Este estudo objetivou avaliar a satisfação de consumidores quanto às condições higiênicosanitárias da alimentação comercializada no mercado público central Isael Almeida de FlorianoPiauí. Foram avaliadas 50 pessoas de ambos os sexos com faixa etária entre 20 e 60 anos que utilizam alimentação do referido estabelecimento, através de aplicação de um questionário semiestruturado composto por 10 itens onde se direcionavam aos pontos mais relevantes da pesquisa. Mais de 30% dos pesquisados respondeu estar insatisfeito com relação à limpeza e arrumação do local onde é servido o alimento. No que diz respeito às instalações sanitárias, 38% referiu estar insatisfeito. Quanto ao conforto oferecido, 32% relatou estar satisfeito e outros 32% também relatou estar insatisfeito. A muita insatisfação no que tange a estética do ambiente foi citada por 40% dos pesquisados. Mais de 25% dos pesquisados mostrou-se muito satisfeito com a variedade e apresentação do cardápio. Com relação a temperatura, sabor e aroma dos alimentos 42% referiu estar satisfeito. Quanto à apresentação pessoal dos funcionários, 30% citou estar satisfeito. Mais de 35% dos estudados relatou estar satisfeito no que diz respeito à simpatia, atenção e educação dos funcionários. Com relação à competência profissional dos funcionários, 32% referiu estar satisfeito. No que diz respeito a rapidez no atendimento, mais de 30% dos pesquisados relatou estar muito satisfeito. Pode-se concluir que mesmo quando ainda há necessidade de intervenção educativa com relação a segurança alimentar, ainda assim há satisfação da maioria dos usuários dos serviços oferecidos. Palavras-chave: Segurança Alimentar. Práticas de Manipulação. Condições Higiênico-Sanitárias. 1Pós-graduada pela Universidade Federal do Piauí e pelo Instituto Federal do Maranhão. Bacharelado em enfermagem pela Faculdade CEUMA. 3Graduanda em Biomedicina pela FACIMP. 4Pós-graduada pelo Instituto Federal do Maranhão. 5Graduação Bacharelado em enfermagem pela Faculdade de Ensino Superior de Floriano- FAESF. 6Graduação em Bacharelado em enfermagem pela Faculdade de Ensino Superior de Floriano – FAESF. 7Graduação Bacharelado em enfermagem pela Faculdade de Ensino Superior de Floriano FAFESF. 8Graduação em enfermagem pela Faculdade de Ensino Superior de Floriano – FAESF 2Graduação Revista Ibero-Americana de Humanidades, Ciências e Educação. São Paulo, v.9.n.09. set. 2023. ISSN - 2675 – 3375 2652 Revista Ibero- Americana de Humanidades, Ciências e Educação- REASE ABSTRACT: Diseases transmitted by water and food are still a serious public health problem in the contemporary world, even with the technological evolution of recent decades regarding food conservation and hygiene techniques, where food is recognized as the main vector of acute enteric illnesses. The hygienic-sanitary conditions of food are of great importance in any environment, as there are many biological, physical and chemical hazards that can cause diseases transmitted by food. Several illnesses can be avoided when food handling is within technical hygiene standards in the pre-preparation and preparation stages. In accordance with the principles of environmental, personal and operational hygiene, the absence of contamination of any origin must be guaranteed. This study aimed to evaluate consumer satisfaction regarding the hygienic-sanitary conditions of food sold in the central public market Isael Almeida de Floriano-Piauí. 50 people of both sexes aged between 20 and 60 years who use food from the aforementioned establishment were evaluated, through the application of a semi-structured questionnaire composed of 10 items which addressed the most relevant points of the research. More than 30% of those surveyed responded that they were dissatisfied with the cleanliness and tidiness of the place where food is served. With regard to sanitary facilities, 38% reported being dissatisfied. Regarding the comfort offered, 32% reported being satisfied and another 32% also reported being dissatisfied. A lot of dissatisfaction regarding the aesthetics of the environment was cited by 40% of those surveyed. More than 25% of those surveyed were very satisfied with the variety and presentation of the menu. Regarding the temperature, flavor and aroma of the food, 42% reported being satisfied. Regarding the personal presentation of employees, 30% said they were satisfied. More than 35% of those studied reported being satisfied with the friendliness, attention and education of employees. Regarding the professional competence of employees, 32% reported being satisfied. Regarding speed of service, more than 30% of those surveyed reported being very satisfied. It can be concluded that even when there is still a need for educational intervention regarding food safety, the majority of users of the services offered are still satisfied. Keywords: Food Safety. Handling Practices. Hygiene and Sanitary Conditions. RESUMEN: Las enfermedades transmitidas por el agua y los alimentos siguen siendo un grave problema de salud pública en el mundo contemporáneo, incluso con la evolución tecnológica de las últimas décadas en cuanto a las técnicas de conservación e higiene de los alimentos, donde los alimentos son reconocidos como el principal vector de enfermedades entéricas agudas. Las condiciones higiénico-sanitarias de los alimentos son de gran importancia en cualquier entorno, ya que existen muchos peligros biológicos, físicos y químicos que pueden provocar enfermedades transmitidas por los alimentos. Se pueden evitar varias enfermedades cuando la manipulación de los alimentos se realiza dentro de las normas técnicas de higiene en las etapas de preparación y preparación. De acuerdo con los principios de higiene ambiental, personal y operativa, se debe garantizar la ausencia de contaminación de cualquier origen. Este estudio tuvo como objetivo evaluar la satisfacción del consumidor con respecto a las condiciones higiénico-sanitarias de los alimentos vendidos en el mercado público central Isael Almeida de Floriano-Piauí. Se evaluaron 50 personas de ambos sexos con edades entre 20 y 60 años que utilizan alimentos del establecimiento antes mencionado, mediante la aplicación de un cuestionario semiestructurado compuesto por 10 ítems el cual abordó los puntos más relevantes de la investigación. Más del 30% de los encuestados respondieron que estaban insatisfechos con la limpieza y el orden del lugar donde se sirve la comida. En cuanto a las instalaciones sanitarias, el 38% manifestó estar insatisfecho. En cuanto a la comodidad ofrecida, el 32% manifestó estar satisfecho y otro 32% también manifestó estar insatisfecho. El 40% de los encuestados mencionó una gran insatisfacción con respecto a la estética del medio ambiente. Más del 25% de los encuestados quedaron muy satisfechos con la variedad y presentación del menú. En cuanto a la temperatura, sabor y aroma de los alimentos, el 42% manifestó estar satisfecho. En cuanto a la presentación personal de los empleados, el 30% dijo estar Revista Ibero-Americana de Humanidades, Ciências e Educação. São Paulo, v.9.n.09. set. 2023. ISSN - 2675 – 3375 2653 Revista Ibero- Americana de Humanidades, Ciências e Educação- REASE satisfecho. Más del 35% de los estudiados afirmaron estar satisfechos con la amabilidad, atención y educación de los empleados. En cuanto a la competencia profesional de los empleados, el 32% afirmó estar satisfecho. En cuanto a la rapidez del servicio, más del 30% de los encuestados afirmó estar muy satisfecho. Se puede concluir que aún cuando aún existe la necesidad de intervención educativa en materia de seguridad alimentaria, la mayoría de los usuarios de los servicios ofrecidos aún se encuentran satisfechos. Palabras clave: Seguridad Alimentaria. Prácticas de Manipulación. Condiciones HigiénicoSanitarias. 1.INTRODUÇÃO A alimentação e a nutrição são requisitos básicos para a promoção e a proteção da saúde. A vigilância nutricional e a orientação alimentar estão inclusas no campo de atuação do Sistema Único de Saúde (SUS), conforme estabelecido na Lei Orgânica da Saúde nº 8.080 de 1990 (JAIME et al., 2018). A luz das rápidas e fortes transformações sociais econômicas que têm caracterizado as economias de muitos países, vêm se observando o aumento da busca pela preferência dos consumidores na maioria dos mercados (ABDALLA, et al., 2012). Além das transformações no cardápio ocorridas nas últimas décadas, houve mudanças profundas na forma de se alimentar, com destaque para os segmentos das populações urbanas que se alimentam fora do domicílio em fast foods e em self-services (OLIVEIRA et al., 2007). A dificuldade de realizar refeições em casa, devido aos longos deslocamentos e a extensa jornada de trabalho, faz com que um grande número de pessoas se alimente na rua (CARDOSO, 2005). O aumento da produção e consumo de alimentos processados é uma das principais causas da atual pandemia de obesidade e de doenças e agravos não transmissíveis (MARTINS, et al., 2013). Uma alimentação saudável, nutricionalmente balanceada, deve ser baseada em alimentos in natura ou minimamente processados (AIN/MP) como raízes e tubérculos, leguminosas, oleaginosas, farinhas, frutas, legumes e verduras, leite, ovos, carnes e pescados (MOREIRA et al., 2023). Garcia (2003) afirma que a forte tendência do crescimento da alimentação fora de casa é favorecida pela diversidade e aumento da oferta de alimentos industrializados de sabor agradável e de custo relativamente baixo o que fica acessível para toda população. Entretanto, este crescimento tem gerado preocupações devido a inadequações nos padrões higiênico-sanitários, necessitando um efetivo controle para reduzir riscos potenciais à saúde do consumidor. Para garantir um controle efetivo, os programas de segurança Revista Ibero-Americana de Humanidades, Ciências e Educação. São Paulo, v.9.n.09. set. 2023. ISSN - 2675 – 3375 2654 Revista Ibero- Americana de Humanidades, Ciências e Educação- REASE alimentar devem abranger todo o fluxo de produção, transporte, armazenamento, manipulação e consumo de alimentos (CAVALLI, 2001). Na década de 80 com a soberania do cliente, com um maior acesso às informações o consumidor passou a exigir novidades, deseja ser tratado como alguém único e ter suas necessidades atendidas e as empresas passaram a ouvir os clientes, o que dá a ele alternativas de escolhas muito maiores (RODRIGUES, 2006). Quando se fala em satisfação do cliente em relação à alimentação, abrange uma série de fatores além da comida oferecida que deve ser de excelência na qualidade acompanhada de um bom atendimento, além de um ambiente agradável. No entanto, muitas vezes, os consumidores não estão preocupados com os aspectos higiênico-sanitários dos alimentos, dos estabelecimentos e dos manipuladores, o que pode vir a conduzir à ocorrência das DTA’s. Os surtos de doenças de origem alimentar geralmente se desenvolvem por falhas como: refrigeração inadequada, preparo do alimento com amplo intervalo (maior que doze horas) antes do consumo, manipuladores infectados/contaminados, processamento térmico insuficiente (cocção ou reaquecimento), conservação a quente imprópria, alimentos contaminados, higienização incorreta, utilização de sobras e uso de produtos clandestinos. O objetivo geral deste trabalho foi avaliar a satisfação de consumidores quanto às condições higiênico-sanitárias da alimentação comercializada no mercado público central de Floriano-Piauí. 2. Materiais e métodos Para a realização desta pesquisa foi utilizado como percurso metodológico, a pesquisa de abordagem quantitativa. 2. 1 O Cenário da Pesquisa O estudo proposto foi realizado no mercado público central Vereador Isael Almeida na cidade de Floriano-PI, entre os meses de junho a agosto de 2022. O mercado Público Municipal Vereador Isael de Almeida conta com diversos boxes com vendas de refeições, bebidas, carnes, produtos hortifrutigranjeiros entre outros. Atualmente o referido mercado encontra-se funcionando em más condições de higiene. Revista Ibero-Americana de Humanidades, Ciências e Educação. São Paulo, v.9.n.09. set. 2023. ISSN - 2675 – 3375 2655 Revista Ibero- Americana de Humanidades, Ciências e Educação- REASE 2. 2 Participantes da pesquisa A presente pesquisa indica como participantes 50 pessoas de ambos os sexos, com faixa etária entre 20 a 60 anos que necessitam se alimentar no mercado central Vereador Isael de Almeida na cidade de Floriano. Os participantes foram selecionados de forma aleatória, visto que, ao chegar ao local da pesquisa aplicou-se o questionário a diversos usuários que por ali circulavam. 2.3 Procedimentos para coleta de dados A coleta das informações necessárias ao desenvolvimento dessa pesquisa se deu por meio de aplicação de 50 questionários com 10 perguntas objetivas com a finalidade de avaliar a satisfação do usuário com relação às condições de higiene do referido estabelecimento. Para Melo e Bianchi (2015), a elaboração das perguntas é etapa crucial para a obtenção de um bom questionário, isto é, de um questionário que cumpra seu papel na coleta de dados de maneira a revelar, da melhor forma possível, a realidade dos fatos ocorridos dentro do alvo do estudo proposto. A pesquisa é do tipo, exploratória que proporciona mais familiaridade com o problema. 2656 3. RESULTADOS E DISCUSSÃO O presente estudo visou avaliar a satisfação de usuários com relação às condições Higiênico–sanitárias da alimentação do mercado público Isael Almeida em Floriano-Piauí. Os resultados obtidos foram apresentados através de tabelas descritivas, foram avaliadas 50 pessoas de ambos os sexos com faixa etária entre 20 a 60 anos. De acordo com a tabela 01, ao serem questionados sobre o grau de satisfação de limpeza e arrumação do local onde é servido o alimento, obteve-se os seguintes resultados: dos 50 pesquisados 32% estão insatisfeitos. Assim há uma necessidade de as autoridades competentes organizarem palestras para as pessoas que trabalham com manipulação de alimentos dentro do mercado central. Tabela 01: Limpeza e arrumação do local onde é servido o alimento Satisfação Nº Muito satisfeito 06 Satisfeito 12 Insatisfeito 16 Muito Insatisfeito 08 Indiferente 08 Total 50 % 12 24 32 16 16 100 Fonte: Dados da pesquisa Revista Ibero-Americana de Humanidades, Ciências e Educação. São Paulo, v.9.n.09. set. 2023. ISSN - 2675 – 3375 Revista Ibero- Americana de Humanidades, Ciências e Educação- REASE A preocupação com a segurança alimentar vem crescendo nos últimos anos, gerando uma série de discussões entre organizações governamentais, instituições de ensino e indústrias alimentícias sobre programas que assegurem à população produtos que não sejam prejudiciais à saúde (RODRIGUES et al., 2003). Considerando que a produção de alimentos com segurança exige cuidados especiais, para que se eliminem quase na sua totalidade, os riscos de contaminação a que esses alimentos estão sujeitos (REZENDE, 2004). Assim, a necessidade de uma ação urgente com a responsabilidade de manipulação dos alimentos dentro dos mercados centrais se faz necessário, para que a população não corra riscos de ingerir alimentos contaminados e venham a ter doenças graves. Em relação a adequação das instalações sanitárias quanto a existência de pias, sanitários, papel toalha e sabonete líquido, observa-se que a maioria dos usuários (38%) estava insatisfeito com as instalações. Tabela 02: Instalações sanitárias (pias, sanitários, papel toalha, sabonete líquido) Satisfação Muito satisfeito Satisfeito Insatisfeito Muito Insatisfeito Indiferente Nº 03 10 19 16 02 % 06 20 38 32 04 Total 50 100 Fonte: Dados da pesquisa Segundo Góes et al. (2004), a limpeza e desinfecção são operações fundamentais, embora muitas vezes feitas de forma inadequada, propicia o desenvolvimento de microrganismos e apresenta um grande potencial de contaminação. A qualidade sanitária dos produtos oferecidos pelos serviços de alimentação configura uma questão fundamental, uma vez que, nesses serviços, as refeições são produzidas em larga escala e tornam-se mais difícil realizar o controle de todas as preparações produzidas, favorecendo o surgimento de doenças transmitidas por alimentos (CARDOSO et al., 2005; ZANDONADI et al., 2007). Nesse sentido, é importante que haja qualidade e um maior controle em relação as instalações sanitárias evitando-se o risco de contaminação dos alimentos. Sobre o conforto oferecido (mesa, cadeira, balcão de atendimento) pôde se observar que 32% dos pesquisados mostraram-se satisfeitos e outros 32% também insatisfeitos. Revista Ibero-Americana de Humanidades, Ciências e Educação. São Paulo, v.9.n.09. set. 2023. ISSN - 2675 – 3375 2657 Revista Ibero- Americana de Humanidades, Ciências e Educação- REASE Tabela 03: Conforto oferecido (mesa, cadeira, balcão de atendimento) Satisfação Muito satisfeito Satisfeito Insatisfeito Muito Insatisfeito Indiferente Nº 01 16 16 12 05 % 02 32 32 24 10 Total 50 100 Fonte: Dados da pesquisa Para uma Unidade de Alimentação e Nutrição (UAN) manter um controle higiênico sanitário eficiente é necessário seguir leis estabelecidas pela Agência Nacional de Vigilância Sanitária (ANVISA). Um dos instrumentos para a qualidade é a elaboração e implantação do Manual de Boas Práticas (MBP) que é fundamental para a produção de alimentos com qualidade, tanto do ponto de vista nutricional quanto em relação à segurança alimentar (ANVISA, 2004). Faz - se necessário trabalhos que visem a melhoria da qualidade dos serviços oferecidos, como palestras, oficinas entre outros. Sobre a estética do ambiente, som, cheiro e estrutura física, observou-se que 40% dos pesquisados se mostrou insatisfeitos com a estética. Tabela 04: Estética do ambiente (som, cheiro e estrutura física). Satisfação Muito satisfeito Satisfeito Insatisfeito Muito Insatisfeito Indiferente Nº 02 07 15 20 06 % 04 14 30 40 12 Total 50 100 Fonte: Dados da pesquisa Conforme Murmann et al. (2007), estabelecimentos limpos, sem indícios de insetos e roedores, que apresentam apenas objetos pertinentes à atividade e com funcionários uniformizados adequadamente demonstram cuidados e aspectos higiênico-sanitários voltados à promoção da saúde do consumidor. Portanto, é importante que a estética do ambiente em que se vai consumir alimentos esteja em condições adequadas e este ganhe simpatia do usuário que consome estes alimentos. Em relação a variedades do cardápio, apresentação dos alimentos 26% dos pesquisados mostrou-se muito satisfeito. Revista Ibero-Americana de Humanidades, Ciências e Educação. São Paulo, v.9.n.09. set. 2023. ISSN - 2675 – 3375 2658 Revista Ibero- Americana de Humanidades, Ciências e Educação- REASE Tabela 05: Variedade do cardápio, apresentação dos alimentos. Satisfação Muito satisfeito Satisfeito Insatisfeito Muito Insatisfeito Indiferente Nº 13 12 07 12 06 % 26 24 14 24 12 Total 50 100 Fonte: Dados da pesquisa O hábito de “comer fora” surgiu de um intenso processo de transformação decorrente do desenvolvimento industrial, que ocorreu na segunda metade do século XX. Hábito este que pode ser analisado de duas perspectivas: a primeira, como atividade social, e a segunda remete ao papel que a mulher passa a desempenhar no mercado de trabalho, contribuindo para um processo de transição nutricional (AKUTSU et al., 2005). Então, nota-se a partir das respostas que os consumidores estão satisfeitos quanto à variedade do cardápio e que aqueles que ofertam produtos variados devem buscar melhoria quanto ao manuseio destes produtos. Em relação à temperatura, sabor e aroma dos alimentos, 42% dos pesquisados mostraram-se satisfeitos. 2659 Tabela 06: Temperatura, sabor e aroma dos alimentos. Satisfação Muito satisfeito Satisfeito Insatisfeito Muito Insatisfeito Indiferente Nº 11 21 04 08 06 % 22 42 08 16 12 Total 50 100 Fonte: Dados da pesquisa As Boas Práticas têm por base o controle das condições operacionais destinadas a garantir a elaboração de produtos seguros. Vale salientar que a adoção das Boas Práticas é requisito fundamental nos serviços de alimentação e a sua correta implantação assegura as condições higiênico-sanitárias dos alimentos, minimizando, assim, os riscos de contaminação (RÊGO et al., 2001). Assim, o manuseio dos alimentos devem ter um rigoroso controle para que sejam elaborados produtos seguros e livres de contaminação e que estes também estejam em temperatura ideal para consumo. Sobre a apresentação dos funcionários quanto ao uso de uniforme, sapatos, toucas e luvas, 30% dos pesquisados mostraram-se insatisfeitos. Revista Ibero-Americana de Humanidades, Ciências e Educação. São Paulo, v.9.n.09. set. 2023. ISSN - 2675 – 3375 Revista Ibero- Americana de Humanidades, Ciências e Educação- REASE Tabela 07: Apresentação dos funcionários quanto ao uso de uniforme, sapatos, toucas e luvas. Satisfação Muito satisfeito Satisfeito Insatisfeito Muito Insatisfeito Indiferente Nº 04 15 10 13 08 % 08 30 20 26 16 Total 50 100 Fonte: Dados da pesquisa Andrade, Silva e Brabes (2003) evidenciam a relação existente entre manipuladores de alimentos e doenças bacterianas de origem alimentar. Podem ser manipuladores doentes, ou portadores assintomáticos, ou que apresentem hábitos de higiene pessoal inadequados, ou ainda que usem métodos anti-higiênicos na preparação de alimentos. É importante que os funcionários estejam uniformizados de forma adequada para que estes manipulem os alimentos de forma adequada, evitando assim a contaminação dos mesmos, evitando assim doenças bacterianas. De acordo com Veiga (2004), não há uma lavagem cuidadosa das mãos antes da manipulação dos alimentos, principalmente após qualquer interrupção e depois do uso dos sanitários. Ao asseio pessoal com boa apresentação, asseio corporal, mãos limpas, unhas curtas, sem esmalte, sem adornos (anéis, pulseiras, brincos, etc.), manipuladores barbeados e cabelos curtos. A utilização de uniforme de trabalho de cor clara é adequada à atividade, mas não é exclusivo para a área de produção. São limpos e em adequado estado de conservação. Sobre a simpatia, atenção e educação dos funcionários, 36% se mostraram satisfeitos em relação a simpatia, atenção e educação dos funcionários. Tabela 08: Simpatia, atenção e educação dos funcionários. Satisfação Muito satisfeito Satisfeito Insatisfeito Muito Insatisfeito Indiferente Nº 13 18 07 04 08 % 26 36 14 08 16 Total 50 100 Fonte: Dados da pesquisa Akutsu et al. (2005) relatam que a padronização do processo de produção de refeição beneficia o trabalho do nutricionista, pois estabelece instruções que possibilitam as operações rotineiras, facilitando assim o treinamento de funcionários, eliminando a Revista Ibero-Americana de Humanidades, Ciências e Educação. São Paulo, v.9.n.09. set. 2023. ISSN - 2675 – 3375 2660 Revista Ibero- Americana de Humanidades, Ciências e Educação- REASE interferência por dúvidas e facilitando o planejamento do trabalho diário. Para o funcionário, esta padronização facilita a execução de tarefas sem a necessidade de ordens frequentes, além de propiciar mais segurança no ambiente de trabalho. A simpatia é importante, pois faz com que o consumidor retorne ao estabelecimento e consuma produtos dos mesmos, visto que quanto mais simpático e atencioso for o funcionário maior clientela terá o estabelecimento que oferece determinado alimento. Em relação à competência profissional dos funcionários 32% dos pesquisados se mostrou-se satisfeito. Tabela 09: Competência profissional dos funcionários Satisfação Muito satisfeito Satisfeito Insatisfeito Muito Insatisfeito Indiferente Nº 12 16 05 07 10 % 24 32 10 14 20 Total 50 100 Fonte: Dados da pesquisa O funcionário responsável pelas atividades de manipulação deve ter capacitação comprovada através de cursos preparatórios com conhecimentos básicos nos temas: contaminantes alimentares, doenças transmitidas por alimentos, manipulação higiênica dos alimentos e boas práticas (BRASIL, 2004). O comprometimento e competência profissional são importantes, para que o consumidor se sinta seguro em consumir alimentos de determinados estabelecimentos e estes se sintam seguros sem riscos de consumirem alimentos contaminados. Em se tratando da rapidez no atendimento ao cliente, observou-se que 34% dos pesquisados se mostraram satisfeitos. Tabela 10: Rapidez no atendimento ao cliente Satisfação Muito satisfeito Satisfeito Insatisfeito Muito Insatisfeito Indiferente Nº 17 15 03 05 10 % 34 30 06 10 20 Total 50 100 Fonte: Dados da pesquisa A rapidez no atendimento ao cliente é algo imprescindível, pois cria laços de confiança entre usuários e estabelecimentos, pois de um modo geral a rapidez no Revista Ibero-Americana de Humanidades, Ciências e Educação. São Paulo, v.9.n.09. set. 2023. ISSN - 2675 – 3375 2661 Revista Ibero- Americana de Humanidades, Ciências e Educação- REASE atendimento sensibiliza o cliente em seu futuro comportamento. Um consumidor insatisfeito com seu tempo de espera talvez possa não desistir naquele momento, entretanto, pode não voltar mais àquele estabelecimento devido à imagem negativa percebida. Poulain (2006) reforça ainda que a alimentação fora de casa desempenha um papel decisivo nas modificações da alimentação na esfera doméstica, no domínio econômico. O atendimento e a rapidez do atendimento são importantes, visto que, a maioria da população opta por se alimentarem fora de casa, devido a praticidade e isto exige daqueles que ofertam seus produtos rapidez no atendimento. CONSIDERAÇÕES FINAIS De acordo com algumas respostas sobre a preocupação dos consumidores com as condições higiênico – sanitárias da alimentação oferecida no mercado público municipal vereador Isael Almeida, muitos estão insatisfeitos mesmo não tendo muita consciência da importância da segurança alimentar. Verificou-se que há a necessidade de intervenção educativa em segurança de alimentos para os manipuladores, é necessário fornecer maiores informações a fim de assegurar um melhor controle higiênico-sanitário dos alimentos no ambiente pesquisado. As Boas práticas de manipulação de alimentos devem ser inseridas, monitoradas e avaliadas periodicamente pelo estabelecimento e pelos órgãos de vigilância sanitária evitando assim DTA’s. A segurança do alimento depende da ação de todos os envolvidos na cadeia produtiva de alimentos desde o produtor da matéria-prima até o consumidor final. Com base nas inconformidades encontradas nos restaurantes e lanchonetes analisadas, buscando assim a melhoria das práticas realizadas no local. Essa pesquisa nos permitiu concluir que há necessidade de constantes vistorias da vigilância sanitária municipal, responsável por promover a saúde na população e prevenir a disseminação de doenças. As irregularidades quanto à ausência de métodos seguros de higienização e preparo dos alimentos reafirma a necessidade da contratação por estes estabelecimentos comerciais, de um profissional nutricionista, que além de criar, possa colocar em pratica, através de capacitação aos vendedores, as formas adequadas de manuseio alimentar. Garantindo Revista Ibero-Americana de Humanidades, Ciências e Educação. São Paulo, v.9.n.09. set. 2023. ISSN - 2675 – 3375 2662 Revista Ibero- Americana de Humanidades, Ciências e Educação- REASE através disso aumento na qualidade e segurança no consumo de alimentos, consequentemente melhorando a satisfação dos consumidores. No âmbito geral, percebeu-se que todos os boxes visitados, estão buscando se ajustar aos padrões exigidos, para garantir a segurança alimentar dos seus consumidores. REFERÊNCIAS ABDALLA, M. M; ALTAF, J. G; TROCCOLI, I. R; TRINTA, J. L. Antecedentesda intenção de recomendação: uma proposição com lanchonetes do tipo fast food. Rev. Bras. Gest. Neg., São Paulo, v. 14, n. 43, p.234-250, abr./jun./2021. ANDRADE, N, J.; SILVA, R. M. M.; BRABES, K. C. S. Avaliação das condições microbiológicas em unidades de alimentação e nutrição. Ciência e Agrotecnologia, Lavras, v. 27, n. 3, p. 590-596, 2003. Disponível em: <http://www.scielo.br/pdf/cagro/v27n3/a15v27n3.pdf>. AKUTSU, R.C; BOTELHO, R.A; CAMARGO, E.B; SÁVIO, K. E.O; ARAÚJO, W.C. Adequação das boas práticas de fabricação em serviços de alimentação. Revista de Nutrição, v. 18, n. 3, Campinas Mai/Jun. 2005. ALVES, F. S., A Organização da Produção de Unidades de Alimentação. ANVISA. Agência Nacional de Vigilância Sanitária. Plantando saúde: Resolução estabelece normas de higiene para alimentos e bebidas á base de vegetais. Revista Saúde Publica. vol. 39, nº 5, 2004. BRASIL. Ministério da Saúde. Agência Nacional de Vigilância Sanitária. Resolução RDC nº 216, de 15 de setembro de 2004. Regulamentos Técnicos sobre de Boas Práticas para Serviços de Alimentação. CARDOSO, R. C. V.; SOUZA, E. V. A.; SANTOS, P. Q. Unidades de alimentação e nutrição nos campi da Universidade Federal da Bahia: um estudo sob a perspectiva do alimento seguro. Revista de Nutrição, Campinas, v. 18, n. 5, p. 669-680, 2005. Disponível em: http://www.scielo.br/pdf/rn/v18n5/a10v18n5.pdf CAVALLI, S. B. Segurança alimentar: A abordagem dos alimentos transgênicos. Revista de Nutrição, Campinas-SP, v. 14 (suplemento), p. 41-46, 2001. GARCIA, R. W. D. Reflexos da globalização na cultura alimentar: considerações sobre as mudanças na alimentação urbana. Revista de nutrição. Campinas, 2003 out/dez.; 16(4): 48392. GÓES, J. A. W.; FURTUNATO, D. M. N.; VELOSO, I. S.; SANTOS, J. M. Capacitação dos manipuladores de alimentos e a qualidade da alimentação servida. Revista Higiene Alimentar, São Paulo, v. 15, n. 82, p. 20-22, 2004. JAIME, O.C; DEMUE, D. C. C; CAMPELLO, T. SILVA, D. O; SANTOS, L. M. P. Um olhar sobre a agenda de alimentação e nutrição nos trinta anos do Sistema único de saúde. Rev Ciência & Saúde, 23(6):1829-1836, 2018. Revista Ibero-Americana de Humanidades, Ciências e Educação. São Paulo, v.9.n.09. set. 2023. ISSN - 2675 – 3375 2663 Revista Ibero- Americana de Humanidades, Ciências e Educação- REASE MARTINS, A. P. B; LEVY, R. B; CLARO, R. M; MOOBARAC, J. C; MONTEIRO, C. A. Participação crescente de produtos ultraprocessados na dieta brasileira (1987-2009). Rev. Saúde Pública, 2013; 47(4); 656-65. MELO, W. V; BIANCHI, C. S. Discutindo estratégias para a construção de questionários como ferramenta de pesquisa. Rev. Brasileira de Ensino de C & T., vol 8, núm. 3, nai-ago. 2015. MOREIRA, C. C; SILVA, T. S; BRITO, F. S. B; OLIVEIRA, A. S. D. Alimentação saudável em encartes de supermercados: refeições segundo a classificação dos alimentos adotada no guia alimentar para a população brasileira. Rev Ciência & Saúde coletiva, 28 (2):631-642, 2023. MÜRMANN, L., SANTOS, M. C., LONGARAY, S. M., BOTH, J. M. C. & CARDOSO, M. 2008. Quantification and molecular characterization of Salmonella isolated from food samples involved in salmonellosis outbreaks in Rio Grande do Sul, Brazil. Brazilian Journal of Microbiology, 39: 529-534. OLIVEIRA, A.C.G.; SPOTO, M.H.F. CANNIATTI-BRAZACA, S.G.; SOUZA, C.W.O.; SOUZA, C.P. Alimentos e Nutrição. 2007, 18 (4):397-403. POULAIN, J. P. Sociologias da Alimentação: os comedores e o espaço social alimentar. Trad. De RPC . Proença, CS Rial, J Conte. Florianópolis: UFSC; 2004. RÊGO, J. C. et al. Proposta de um programa de boas práticas de manipulação e processamento de alimentos para unidades de alimentação e nutrição. Revista Higiene Alimentar, São Paulo, v. 15, n. 89, p. 22-27, 2001. REZENDE, C. Estafilococcos em Alimentos: significado para a Saúde Pública, Revista Higiene Alimentar, v.18, n.122, p.32-37, julho 2004. RODRIGUES, K. L.; GOMES, J. P.; CONCEIÇÃO, R. C. S. da; BROD, C. S.; CARVALHAL, J. B.; ALEIXO, J. A. G. Condições higiênico-sanitárias no Comércio Ambulante de Alimentos em Pelotas-RS. Revista Ciência e Tecnologia de Alimentos, Campinas, 23(3), p.447-452, set-dez 2003. RODRIGUES, R. D. Unidade de Alimentação de Nutrição. Revista CRN – 3, São Paulo, nº 83, jul/Ago/Set. 2006, p.15. VEIGA, C. F.; DORO, D. L. OLIVEIRA, K. M. P.; BOMBO, D. L. Estudos das condições sanitárias nos estabelecimentos comerciais de manipulação de alimentos no município de Maringá, PR. Higiene Alimentar, jan./jun., vol. 20, nº 138, p. 28-36, 2004. ZANDONADI, R. P. et al. Atitudes de risco do consumidor em restaurantes de auto serviço. Revista de Nutrição. Campinas-SP, v. 20, n. 1, p. 19-26, 2007. Revista Ibero-Americana de Humanidades, Ciências e Educação. São Paulo, v.9.n.09. set. 2023. ISSN - 2675 – 3375 2664
https://openalex.org/W2102765339	https://www.macrothink.org/journal/index.php/ijafr/article/download/3372/2985	English	null	How CFO’s communicate financial information - A pedagogical task in theory and practice	International journal of accounting and financial reporting	2,013	cc-by	9,627	International Journal of Accounting and Financial Reporting ISSN 2162-3082 2013, Vol. 3, No. 1 Received: March 12, 2013 Accepted: April 07, 2013 DOI: 10.5296/ijafr.v3i1.3372 eived: March 12, 2013 Accepted: April 07, 2013 DOI: 10.5296/ijafr.v3i1.3372 How CFO’s communicate financial information - A pedagogical task in theory and practice Krister Bredmar School of Business, Economics and Law, University of Gothenburg Box 610, S-405 30 Gothenburg, Sweden E-mail: krister.bredmar@gu.se Krister Bredmar School of Business, Economics and Law, University of Gothenburg Box 610, S-405 30 Gothenburg, Sweden E-mail: krister.bredmar@gu.se 1. Introduction Each profession has its own concepts and ways of working. It is often described as expertise or experience. In most large organizations, there is a professional group of accountants who largely have their own language, and even live by their own logic, the economic rational. In many cases, they work with a financial mirror of the activities, a mirror to help various decision makers in the organization to better understand what is happening in the business and how it can be adjusted to work even better. To achieve this, the accountant has an obligation to communicate the financial conditions in a clear and meaningful way. This challenge, which can be described as a practical challenge, is the core of the research question that this study is based on (Booth, Colomb, & Williams, 1995). If the aim is to understand the pedagogical communication of financial information, the accountant becomes a logical and natural starting point. This study describes the pedagogical communication of financial information from two perspectives. First, from a theoretical perspective, focusing on how it should be designed, and secondly from a practical perspective where financial managers in Swedish municipalities are asked to respond to propositions about how they work with communication. The accountant is facing a major challenge, to learn to communicate financial information as pedagogical as possible, especially in the light of the power that is imbedded in the financial language (Rombach, 2005). Pedagogical activities of various kinds occur in virtually all-social contexts (Trenholm & Jensen, 2000). This form of pedagogical practice is particularly obvious in a school context where the contextual conditions require a teacher and a student. But pedagogical activities are also present in a form of pedagogical practice between doctors and patients, architects and supervisors or middle managers in general and their colleagues (Bernstein, 2000). In a social context, the cultural and social events and phenomena are produced and reproduced (Macintosh, 1994). Basil Bernstein is a British sociologist interested in these issues in general and how pedagogical communication can be described in particular. One of his most important starting points is that pedagogical communication occurs in a social context, in what he calls a pedagogic discourse (Bernstein, 2000). He sees the text as a carrier of content that is related to a certain time and a certain context. This becomes the starting point for how pedagogical activities can be understood in a social context. Abstract An important result of communication is to gain a deeper understanding, and for the receiver to create meaning of that which is communicated by the transmitter. In a way, this can be described as the core of pedagogical communication. The premise of this study is that pedagogical communication can be understood and explained by means of financial reports (1), oral presentations (2), concepts and illustrations (3) and by the social context, the group where the communication takes place (4). The theoretical areas that form the basis of the first question in this study, how the communication can be designed, are based on these four areas. In a second part, 111 CFOs in Swedish municipalities have responded, via a web-based questionnaire, how they work with these areas. The result shows that they are largely working with several of the pedagogical areas, such as the financial reports and oral presentations, but that there are also areas such as the social context and the group's importance in the pedagogical communication that are not as developed. Although much is already working well, there are areas that can be developed to get the pedagogical communication of financial information to work even better. Keywords: Pedagogical communication, Swedish municipalities, financial managers 162 www.macrothink.org/ijafr International Journal of Accounting and Financial Reporting ISSN 2162-3082 2013, Vol. 3, No. 1 www.macrothink.org/ijafr International Journal of Accounting and Financial Reporting ISSN 2162-3082 2013, Vol. 3, No. 1 International Journal of Accounting and Financial Reporting ISSN 2162-3082 2013, Vol. 3, No. 1 complex, even though the language does not make the context less complex, just more graspable. The ability to communicate in a financial area is a skill that is increasingly valued by both teachers at universities and industry (Abdullah Zaid & Abraham, 1994; Andrews & Koester, 1979, Kavanagh & Drennan, 2008; Morgan, 1997). Through language different meanings are shaped, and the social context is produced and reproduced (Rönnström, 2006). According to Bernstein's (2000) models, language represents an important function used to transfer meaning through communication. But he also describes this transfer as a pedagogic function where a potential pedagogical meaning is transmitted, which is the purpose of pedagogical communication. The language itself is not free from meanings and values, which may be transferred, in a pedagogical communication, but the language is controlled by certain rules, which, for example, become clear in an argument (Toulmin, 2003). A certain rhetorical meaning or a way to build up the various claims makes the language the carrier of intention and not just opinion. From another perspective, one can also use language to create or produce an understanding, something, which Weick (2001) calls "enactment." The meaning or sense of what is communicated is created by the one who communicates, and the receiver benefits from the meaning. Language is thus not only a carrier of a given meaning, but also an understanding or meaning, an interpretation. In a social context, that Schön call practice, communication is often a purely intuitive and almost automatic reflection (Schön, 1983). The reflection is not just about a given problem or situation, but also becomes a way to orient oneself, where the understanding of oneself becomes important. This understanding of self or the organization can then be communicated in different ways, transferred to others in a form of organizational sense making (Checkland & Holwell, 1998). The ability to communicate in a pedagogical way becomes crucial in order to transfer an understanding of the organization, and to develop the social context (Habermas, 1995). For decision makers in large organizations to be able to understand what is going on in the operations, if changes are needed, and if business is good or bad, a working financial reporting is needed. This is the accountants’ responsibility, especially when it comes to compile financial information, process it and then spread it. 1. Introduction Language is also an important tool in a social context (Bickerton, 1995). The language's smallest parts, words or concepts are different forms of knowledge banks or stored meanings (Berger & Luckmann, 1991). Language thus becomes an interface where a common understanding and an ability to translate meaning are central (Hasan, 1992). Through language, everyday life becomes objectified and possible to communicate, and experiences that are hard to put into words become hard to communicate a meaning about (Berger & Luckmann, 1991). Although language was largely about face-to-face communication from the beginning, you can also use language as a basis for written communication. In some areas and disciplines, language is an expression of power and control (Bernstein, 2000). One such example is the financial language (Rombach, 2005). In many contexts, the financial language becomes a form of rational rhetoric in which emotions and irrational arguments do not bite. The financial language also simplifies an organizational context, which actually is very www.macrothink.org/ijafr 163 2. Theoretical background Theories about communication in general, and communication of financial information in particular, basically describe how certain information has been compiled and then transferred from a transmitter to a receiver. Pedagogical communication is largely based on how a deeper understanding and meaning is achieved (Bernstein, 2000; Checkland & Holwell, 1998; Hines, 1988; Weick, 2001). This communication occurs in different types of information networks where different actors ensure that the right information reaches the right individual, for example, depending on the function or position s/he represents in an organization (Macintosh, 1994). There are four different areas interesting to study, and these can also be used to search for a deeper understanding of the pedagogical communication of financial information. The first area concerns how the reports are compiled, which can also be described as the written documentation. The second area concerns how the oral communication works. The third area has the concepts and illustrations in focus, and the last area is about how communication works in a group. Theories in these four areas are presented in this chapter, and they form the basis for the propositions that the survey is based on. An important point in this chapter is that communication takes place in a context, i.e., in the organization, and that an understanding of reality is created and re-created with the help of the financial information that is communicated (Hines, 1988; Macintosh, 1994). This means that a form of discursive analysis is done where the communication phenomenon is studied from a financial managerial perspective in a given context, which is the municipality he/she is engaged in. All propositions and theoretical ideas are not directly linked to the discursive approach. It is still an interesting method and an important perspective to use to try to catch a deeper dimension of what is going on in an organization in general and how financial information is communicated in particular. "In this chapter, discourse is viewed as a way of knowing or a perspective for understanding organizational life. It is a lens or a point of entry for seeing, learning and understanding ongoing events. As a lens, it provides a unique way to focus on the subtle aspects of organizing and to determine what is figure and ground in the framing of organizational events. "(Putnam & Fairhurst, 2001, p. This is a common and classic approach to information management (Alter, 2002). With the increasingly extensive and complex information systems, the ability to compile and disseminate information has become more critical and central to large organizations. It is thus necessary to be able to communicate in a pedagogical way, and in this study, the aim is to try to understand how this communication can be developed further and become a more pedagogical communication. In light of the reasoning, it is interesting to study how accountants and CFO’s communicate financial information and meaning to the organization. The aim of this study is to clarify how a pedagogical communication can be described and understood in organizations in order to analyze and understand the meaning pedagogical communication contributes to a social context. 164 www.macrothink.org/ijafr www.macrothink.org/ijafr International Journal of Accounting and Financial Reporting ISSN 2162-3082 2013, Vol. 3, No. 1 International Journal of Accounting and Financial Reporting ISSN 2162-3082 2013, Vol. 3, No. 1 2. Theoretical background 79) In the discourse used, the financial language becomes a base for communication, and this is an important part of understanding how successful pedagogical communication can be described and analyzed. The financial language is full of concepts and expressions that distinguish it from the everyday language used in the community. In the organization, the financial and accounting language become an important part of the pedagogical communication, and how the language is used becomes a way of understanding the finance and accounting function and its role in the organization. Each section in the following chapter ends with one or more propositions, which are later used in the survey. The proposition is meant to be a kind of summary of the theoretical outline. In the questionnaire, the respondent should answer the proposition with a number on a scale of 1 to 7, where 1 means that he/she does not agree with the proposition, while 7 means that the respondent fully agrees with the proposition. www.macrothink.org/ijafr www.macrothink.org/ijafr 165 2, The structure of the written financial reports is largely tailored to the recipient, and we rarely use general statements. The information compiled and communicated is often the basis for various kinds of decisions in an organization (Alter, 2002). In its simplest form, this is done automatically in the systems controlled by machines, but in most cases decisions include some form of human intervention. When a decision maker has to choose between two or more alternatives there is usually also a need for some kind of judgment or interpretation of an analysis. In order to identify what the options are, the ones involved need to define and demarcate the area that decision affects, and the consequences and effects of the decision. The information communicated creates opportunities to evaluate various options and the effects that the options will generate. An important function and a common use of the financial information are as the basis for analysis and decision, as the third argument focuses on. 2.1 Financial reports - the written documents 2.1 Financial reports - the written documents In a large organization like a municipality, financial reporting is largely based on the reports produced in the computerized information systems. These are usually based on the accounting. They are a form of codes (Putnam & Fairhurst, 2001) that represent what has happened in the business and are the basis for financial reporting. But to succeed in the pedagogical communication of financial information there is also a need for non-numeric reporting. Andrews and Koester (1979), for example, emphasise "the significance of these nonnumerical reports". To work with a more active reporting, it is important that the financial reporting is supplemented with a non-financial/non-numeric reporting. This is the starting point for the first proposition. 1, In our organization, we are equally committed to communicating financial information both in figures and in written text. Another way to understand how financial communication works in an organization is based on the structures communication and reports are based on (Putnam & Fairhurst, 2001). In many cases, a given structure from the report templates are used, but it may also be that the structure has been adapted to the receiver in order to focus on and emphasize the information that is most important for that particular recipient. Here, the value of communication is increased through an adjustment of the report structure rather than a use of the general statements. This is the starting point for the second proposition. 2, The structure of the written financial reports is largely tailored to the recipient, and we rarely use general statements. International Journal of Accounting and Financial Reporting ISSN 2162-3082 2013, Vol. 3, No. 1 2.1 Financial reports - the written documents International Journal of Accounting and Financial Reporting ISSN 2162-3082 2013, Vol. 3, No. 1 3, The written financial reporting is very common as a basis for analysis and decision. The written financial reporting is very common as a basis for analysis and decision. "Because people need information to participate in economic life, the extensive use of computerized information sources ... raises questions about whether technical progress will divide the haves and have-nots in society even further." (Alter, 2002, page 297) "Because people need information to participate in economic life, the extensive use of computerized information sources ... raises questions about whether technical progress will divide the haves and have-nots in society even further." (Alter, 2002, page 297) A requirement to conduct the daily work in many organizations is to have access to appropriate information. The need for financial information and communication can be understood from the responsibility a particular recipient of the information has. Access to the information is then controlled by the task or work the receiver does, a constraint of the www.macrothink.org/ijafr 166 www.macrothink.org/ijafr 2.2 The presentation - oral communication As the language is an important part of communication in a social context it also becomes an interesting dimension to study (Taylor, 1999). Communication can then be understood in part as a set of rules or procedures that supports communication and in part as different stories and narratives that create the organization's identity (Browning, 1992). With the help of the narratives and the stories, an organizationally rooted knowledge is created in a group. As a first step, it is important that it is done at all, which is the basis for the following proposition. 5, We are used to present (we present regularly) financial information verbally. International Journal of Accounting and Financial Reporting ISSN 2162-3082 2013, Vol. 3, No. 1 International Journal of Accounting and Financial Reporting ISSN 2162-3082 2013, Vol. 3, No. 1 dissemination of information, basically communication (Alter, 2002). A form of physical control of communication can also be done with passwords or restrictions on the locations where the information is available. It is common to this type of analysis of the availability of information that those who work in a particular area or a particular part of the business should have access to information, which is the basis for the fourth proposition. 4, The financial information is easy to access for those who work in our organization. 4, The financial information is easy to access for those who work in our organization. 2 2 The presentation - oral communication 2.2 The presentation - oral communication 5, We are used to present (we present regularly) financial information verbally. One way to understand the communication is that it evolves in two steps, where a translation is made (Taylor, Cooren, Giroux, & Robichaud, 1996). First, a sentence from a report is translated into a conversation, a written text transformed to talk, and in the next step the conversation is translated into text. With the help of this translation process, an organizational identity is created, in the complex network of people that the organization represents. Since it is important that a meaning is transmitted, it is natural to consider how a message is perceived. This is particularly central in a financial context where meaning is strongly linked to an activity and may have implications for decision. Transmission of meaning can also be understood as something that is non-verbal, i.e. where one message and meaning is reinforced in a particular situation and social context, which is sometimes part of a more elusive power language. Abdullah Zaid and Abraham (1994) describe it as follows. "Such a framework may be particularly relevant to accounting because consideration would be given to the accuracy of the transmission, the exactness with which the meaning of the transmission is conveyed and the extent to which the conveyed meaning produces the desired conduct, thus allowing an increased concentration on non-verbal communication skills. " This reasoning is the basis for the following two propositions. 6, In our organization we often choose to present financial information verbally because then we can see if the receiver understands what we want to communicate. 7, When the financial information has been presented, it is important for us to listen to questions and try to clarify the meaning of the information, if there is anyone who did not understand the meaning of the message. With the help of oral communication the sender may also use a more casual language, which in itself makes a message or a meaning more accessible. This is especially important when there is a meaning that should be communicated (Bernstein, 2000), and when there is an www.macrothink.org/ijafr www.macrothink.org/ijafr 167 ISSN 2162-3082 2013, Vol. 3, No. 1 understanding that needs to be created (Weick, 2001). This is the starting point for the next proposition. understanding that needs to be created (Weick, 2001). This is the starting point for the next proposition. 8, When we present financial information orally, we always try to use a language that is accessible and does not make it harder for the recipient to understand the meaning of information. 9, Our reporting is based largely on communicating a meaning, a concept, rather than just numbers. 9, Our reporting is based largely on communicating a meaning, a concept, rather than just numbers. The financial language in itself is an important means of communication. In international organizations, it may even be perceived as more important than English (Macintosh, 1994). The financial language can also be a form of power language as it is difficult to argue with someone who uses financial and accounting concepts in an argument (Rombach, 2005). It is therefore important that the person who receives a report is aware of the concepts that make up the structure of the report, which the next proposition is about. 10, The recipient is fully aware of the meaning of the concepts we use in reporting. 10, The recipient is fully aware of the meaning of the concepts we use in reporting. As the business administration methods often have an inherent structure, which, for example, becomes clear in a financial statement, it is natural to use that structure in the regular reporting (Frenckner, 1982, Smith, 2006). But it is also important that the message communicated is amplified by the structure used, and the way that the message is organized (Andrews & Koester, 1979). A suitable structure is not necessarily better or worse than a standard structure as long as the structure supports the purpose of the communication. This is the basis of the following proposition. 2.3 The content of the communication - concepts and illustrations Financial reporting and communication is largely characterized as being about content and meaning. But this does not exclude that the form of communication is important. A core competence of an accountant is the ability to formulate a concept and thus a meaning (Andrews & Koester, 1979). The concepts are means to convey and communicate a deeper meaning, where for example a budget is not just a compilation of numbers but also an expression of responsibility and authority (Emmanuel, Otley, & Merchant, 1990). This ability is the basis for the next proposition. 11, When financial information is communicated it is to a large extent based on a well thought out structure and with a clear purpose. 11, When financial information is communicated it is to a large extent based on a well thought out structure and with a clear purpose. Using illustrations such as graphs, more dimensions can be added to what is communicated (Moriarity, 1979). In many cases, the report can be a graphical interface where the recipient receives a deeper understanding by using illustration (Alter, 2002). The following proposition is about this. 12, Our reports often contain graphic illustrations of the financial information, such as charts. 168 www.macrothink.org/ijafr 16, The financial information is largely adapted to the group that receives the information. With the help of these four dimensions, financial reports, oral presentation, communication content and communication to a group, this study focuses on areas that enable and clarify how financial information can and to some extent should be communicated. This becomes a form of operationalization of an area that could be perceived as vague and that could be studied from many perspectives. There is still much to be done in the field of pedagogical communications and communicative action (Englund, 2007), where this schematic description of the four areas is a way to understand the field. 15, When financial information is communicated, we are very keen on communicating its meaning and not just a number or a report. If the group will use the information for decision making or as part of an evaluation of its own operations, it is also important that the information is adapted to the context in which the group is in, the reality that the financial information helps to create (Hines, 1988). The information may for example involve that only the figures relating to the group's operations are presented. This is illustrated by the following proposition. International Journal of Accounting and Financial Reporting ISSN 2162-3082 2013, Vol. 3, No. 1 2.4 Communicating together – the role of the group One way of analysing financial information is with the help of a work team or a part of an organisation. This gives the group a very important role in creating a common understanding of what is reported (Taylor et al., 1996), where the group or organization becomes part of a process creating meaning (Checkland & Holwell, 1998). The ability to create contexts and meetings where a meaning is developed is an important part of the pedagogical communication (Taylor, 1999), and this forms the basis of the following two propositions. 13, It is often the case that we communicate financial information to a group working on a particular task, which means that they can interpret the meaning of the information together. 14, In our organization, it is common to discuss the financial information reported to each other in order to understand what it means. The group creates an understanding or meaning based on the translation of texts that is done in the group and by the narratives that create the identity of the group, the organization (Browning, 1992; Checkland & Holwell, 1998; Taylor et al., 1996). Together, each member of the group contributes to "interpret the language of numbers into understandable information" (Abdullah Zaid & Abraham, 1994). This process thus dealswith the meaning rather than the numbers to a very large extent (Taylor, 1999; Taylor et al., 1996), which the next proposition is about. 15, When financial information is communicated, we are very keen on communicating its meaning and not just a number or a report. International Journal of Accounting and Financial Reporting ISSN 2162-3082 2013, Vol. 3, No. 1 different stakeholders, and thus are in major need of communicating financial information. The CFO is responsible for communicating financial information in an efficient and pedagogical manner. With the ambition to undertake a broader study follows the intent to try to describe a broader picture in which more general observations are presented and analyzed (Holme & Solvang, 1991; Rienecker & Jorgensen, 2008). A natural consequence, given this initial position, is that material needs to be collected using a method suitable for this, especially from an efficiency perspective. The method chosen was a survey with standardized questions where comparable responses were collected (Fowler, 1995). Since the aim is to use questions based on deductive thinking this was the most appropriate method. The questions are formulated as statements, which respondents are asked to relate to, using a Likert scale (Clason & Dormody, 1994). The statements are a form of operationalization of the theoretical foundations that the study is based on. If the respondent answers 1 on the Likert scale it means that he/she agrees to a very small extent with the statement, while the answer 7 means that he/she agrees to a large extent. The empirical data, which a survey method is based on, can be collected in different ways. The most classic is the use of a questionnaire, which is distributed through mail. In this study, an email with a link to a web page, which lead the respondent to a questionnaire, was sent to the CFO´s. The email sent with the cover letter was directed to "The CFO” in the title, which meant that the email was forwarded to the municipal finance officer. The questionnaire was compiled in the Google tool surveys, and the link between the answers in the questionnaire and the underlying Excel sheet was handled by Google’s homepage. The questionnaire includes 20 questions and propositions where four questions are about the municipality that the respondent works in. The remaining propositions are formulated using the theoretical areas, where each area is represented with two questions. The questionnaire for this study represents a suitable form, and using the web interface is a way of collecting data in an efficient way. 111 CFOs out of the 290 contacted responded, giving a response rate of 38%. 3. Method This study is about how CFO’s in Swedish municipalities are working to communicate the financial information in a pedagogical way. It is a survey (Bryman & Bell, 2005; Holme & Solvang, 1991) in which all 290 Swedish municipalities were included, and CFO’s in all municipalities were contacted. Municipalities are interesting as they work with so many www.macrothink.org/ijafr 169 www.macrothink.org/ijafr This response rate is above the 36.83% that Sheehan’s (2001) study showed as the average for this type of web surveys. This means that this study is neither better nor worse than comparable studies, but follows average. 4. Results The empirical material gathered through the survey is initially grouped according to the four theoretical perspectives. In this first part, a description based on the distribution of answers per question is interesting along with the mode values that can be identified (Bystrom, 1990; Eriksson, 1978). This is followed by a section based on an attempt to weigh the answers in average, despite the fact that a Likert scale is not really suitable for this. 4.1 Financial reports - the written documents The four propositions which form the basis for the financial reports category show a relatively high and equal distribution among the three next highest alternatives, 4, 5 and 6, as seen in the table below. One observation that perhaps stands out is that 5 respondents answered 2, i.e. to a small extent agree with the second contention. This could mean that they use more general reports and few costumed reports. Another observation worth noting is that as many as 16 respondents answer with the highest response, 7, in terms of communicating www.macrothink.org/ijafr 170 www.macrothink.org/ijafr International Journal of Accounting and Financial Reporting ISSN 2162-3082 2013, Vol. 3, No. 1 financial information both in numbers and in written text. Even to question 3 there is a large number, 10 respondents with the number 7 on the proposition that they use financial reports as a basis for analysis and decision. financial information both in numbers and in written text. Even to question 3 there is a large number, 10 respondents with the number 7 on the proposition that they use financial reports as a basis for analysis and decision. Table 1. Answers to proposition 1 to 4 Table 1. Answers to proposition 1 to 4 Proposition 1 2 3 4 5 6 7 1, In our organization, we are equally committed to communicating financial information both in figures and in written text. 1 6 10 37 40 16 2, The structure of the written financial reports is largely tailored to the recipient and we rarely use general statements. 1 5 19 32 29 22 2 3, The written financial reporting is very common as a basis for analysis and decision. 1 2 13 32 49 10 4, The financial information is easy to access for those who work in our organization. 3 8 20 38 36 5 The most common answer that is the mode value for the first question is 6, indicating that a large proportion of the respondents answered that. This is similar for the third question, where 49 respondents, representing 46% of the respondents, indicated value 6 on a Likert scale. Thus, a large proportion agrees with the statement. For the second and fourth questions, e the distribution is more even among several alternatives. The second question’s mode value was 4, marginally before 5, and for the fourth question the mode value was 5 with an even smaller difference to 6. Overall, the propositions were answered with high responses, with a slightly more even distribution of question 2 and 4. 4.2 The presentation - oral communication The distribution between the different responses in the second theoretical group is shown in Table 2 below. What can be observed is that the respondents, to a very large extent, present the financial information verbally (proposition 5), but there are also a number of respondents who differ and say they almost never present information orally. Although the sixth 171 www.macrothink.org/ijafr International Journal of Accounting and Financial Reporting ISSN 2162-3082 2013, Vol. 3, No. 1 proposition has a relatively high but slightly more even distribution around 5, responses indicating that the respondents are dealing with the ambition that the recipient should understand the information. However, virtually all state that they listen to questions, proposition 7, which in itself is an interesting observation since there is a group of respondents who do not present orally. Even the eighth proposition has a relatively high distribution and lacks answers in the lowest response options. The respondents seem to work actively to use an easy-to-understand language so that the recipient should understand what is communicated. They also agree to try to answer questions to clarify if there is anyone who listened but did not understand the message. Table 2. Answers to proposition 5 to 8 The mode value for the eighth proposition is 6, answered by 51 respondents, that is, they u an easily accessible language. Around the mode value for proposition 7 and 6, and in som Proposition 1 2 3 4 5 6 7 5, We are used to present (we present regularly) financial information verbally. 1 2 2 4 22 44 35 6, In our organization we often choose to present financial information verbally because then we can see if the receiver understands what we want to communicate. 2 4 6 20 46 27 4 7, When the financial information has been presented, it is important for us to listen to questions and try to clarify the meaning of the information, if there is anyone who listened that did not understand the meaning of the message. 4 12 28 43 22 8, When we present financial information orally, we always try to use a language that is accessible and does not make it harder for the recipient to understand the meaning of information. 2 3 10 31 51 13 Table 2. Answers to proposition 5 to 8 The mode value for the eighth proposition is 6, answered by 51 respondents, that is, they use an easily accessible language. Around the mode value for proposition 7 and 6, and in some sense also 5, there is a clear distribution, almost shaped like a normal distribution. Proposition 6 has mode value 5, and proposition 7 and 5 have mode value 6. The mode value for the eighth proposition is 6, answered by 51 respondents, that is, they use an easily accessible language. Around the mode value for proposition 7 and 6, and in some sense also 5, there is a clear distribution, almost shaped like a normal distribution. Proposition 6 has mode value 5, and proposition 7 and 5 have mode value 6. The mode value for the eighth proposition is 6, answered by 51 respondents, that is, they use an easily accessible language. Around the mode value for proposition 7 and 6, and in some sense also 5, there is a clear distribution, almost shaped like a normal distribution. Proposition 6 has mode value 5, and proposition 7 and 5 have mode value 6. www.macrothink.org/ijafr 172 www.macrothink.org/ijafr International Journal of Accounting and Financial Reporting ISSN 2162-3082 2013, Vol. 3, No. 1 4.3 Content of the communication - concepts and illustrations The third group of propositions has a relatively even distribution between several of the responses, as seen in the table below. The majority of respondents, for example, answered 5 or 6 with a normal distribution similar frequency of responses 4 and 7. A slightly clearer normal distribution can be seen for the responses to proposition 10, which is centered on 5. Proposition 11 and 12, however, are more evenly distributed. The first two propositions, whether a meaning is communicated and if the recipient is aware of the implications, show a more normally distributed frequency, indicating that respondents are quite unified in their opinions, while the responses vary more in terms of whether they use a clear structure and if they use graphic illustrations. Table 3. Answers to proposition 9 to 12 Table 3. Answers to proposition 9 to 12 Table 3. Answers to proposition 9 to 12 Proposition 1 2 3 4 5 6 7 9, Our reporting is based largely on communicating a meaning, a concept, rather than just numbers. 2 8 14 39 36 12 10, The recipient is fully aware of the meaning of the concepts we use in reporting. 4 8 25 46 19 4 11, When financial information is communicated it is to a large extent based on a well thought out structure and with a clear purpose 1 1 5 14 39 43 6 12, Our reports often contain graphic illustrations of the financial information, such as charts. 1 6 10 15 30 38 10 It is really just for the tenth proposition with a clear mode value, that it is meaningful to talk about a traditional mode value. 46 respondents answered 5 on the proposition if the respondent is well aware of the meaning of the concept being communicated. For the other three propositions, there are even distributions between 5 and 6. This is also high, even slightly higher than in the tenth proposition, but it indicates a broader view than what is shown with a mode value. 4.4 Communicating together – the role of the group The fourth and last group of propositions shows the greatest variation compared to the previous groupings, with one exception. The fifteenth proposition, whether it is a meaning that is communicated, has a large, clear percentage that answered 5. In the other propositions, 173 www.macrothink.org/ijafr www.macrothink.org/ijafr 4.5 A more general description Since the survey and propositions are based on a Likert scale it is not suitable to calculate mean and standard deviation. But in trying to illustrate and communicate the results in a pedagogical way, means and standard deviations were calculated for the various propositions and then presented in a radar chart. The results of the calculations are in line with the previously reported results. The responses are relatively even and high with fairly centered spread. The calculation is shown in the table below. Table 5. Mean and standard deviation for each proposition Proposition nr 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Mean 5,42 4,43 5,45 5,01 5,87 4,84 5,61 5,5 5,22 4,75 5,22 5,01 4,58 4,89 5,55 4,91 Standard-deviation 1,11 1,22 0,99 1,11 1,16 1,19 1,04 1,03 1,14 1,08 1,06 1,36 1,26 1,12 1,04 1,18 Table 5. Mean and standard deviation for each proposition If one were to analyze this calculation into three groups, the first would include propositions that the respondents are working a lot with and has experience from. Here we find high averages with fairly tight standard deviations suggesting a narrow normal distribution. The propositions that fall within this group is 1, to communicate both in figures and in written text, 3, the financial report is the basis for analyzes and decisions, 5, people are used to present the information orally, 7, listening to questions, 8, using an easy-to-understand language and 15, they want to communicate a meaning. An interpretation of the answers could be that these are areas that they have developed routines and ways of working with. The group of propositions that would be the opposite is the ones that have lower numbers as weighted responses. 4 means a position in the middle of a Likert scale, and it can in a way be understood as if they neither agree nor oppose the proposition. The propositions that have the lowest weighted average is 2, the structure of the reports are customized, 6, to present orally to ensure that the receiver has understood, 10, the recipient is aware of what the concepts mean, 13, 14 and 16 about the group interpreting the information together. These are areas that one could possibly interpret as having room for development, especially in terms of how groups work with the information together. International Journal of Accounting and Financial Reporting ISSN 2162-3082 2013, Vol. 3, No. 1 the respondents have a slightly wider and more diverse understanding where the answers vary evenly between the numbers 4, 5 and 6. The three claims are more clearly linked to how the group interprets and manages the information, indicating that this is solved in different ways in the studied organizations. It is also clear that only the mode value of the fifteenth proposition is meaningful to highlight. In the other propositions, the difference between the responses is too small, which means that the mode value is misleading. p g Table 4. Answers to proposition 13 to 16 Proposition 1 2 3 4 5 6 7 13, It is often the case that we communicate financial information to a group working on a particular task, which means that they can interpret the meaning of the information together 2 4 13 26 37 18 5 14, In our organization, it is common for us to discuss the financial information reported to each other in order to understand what it means 1 3 7 23 41 32 3 15, When financial information is communicated, we are very keen on communicating it's meaning and not just a number or a report 1 1 14 27 51 15 16, The financial information is largely adapted to the group that receives the information 1 3 7 26 36 31 6 An overview based on the results could be described as follows: CFO’s in the studied municipalities operate in a satisfactory way with the written documents and oral presentations, while there are slightly lower response distributions and greater variation in terms of how Table 4. Answers to proposition 13 to 16 Table 4. Answers to proposition 13 to 16 An overview based on the results could be described as follows: CFO’s in the studied municipalities operate in a satisfactory way with the written documents and oral presentations, while there are slightly lower response distributions and greater variation in terms of how they work with concepts and illustrations, and especially how they use the group to interpret what is communicated. It is also interesting that respondents generally respond with relatively high responses, and that in many cases there are natural normal distributions on the responses, www.macrothink.org/ijafr www.macrothink.org/ijafr 174 indicating that the responses are focused. There is no proposition that has a majority of low responses. All propositions had the majority of the answers to the top half, which means that the CFO wants to communicate the financial information in a pedagogical way. 4.5 A more general description The remaining four propositions end up right in between what works well and what can be developed. 175 www.macrothink.org/ijafr www.macrothink.org/ijafr International Journal of Accounting and Financial Reporting ISSN 2162-3082 2013, Vol. 3, No. 1 Figure 1. Illustration of mean for each proposition International Journal of Accounting and Financial Reporting ISSN 2162-3082 2013, Vol. 3, No. 1 Figure 1. Illustration of mean for each proposition One possible way of illustrating mean could be in a radar chart. Every mean from each proposition is placed on one of the lines in the radar chart. Within the chart there is also a possible classification where the first four propositions belong to financial reports. Here we have an even distribution except for proposition 2, which is slightly lower. In the second classification or category, propositions 5-8 are found, and this group also has a high mean with a slightly lower mean of the sixth proposition. The third classification includes proposition 9 to 12, and here we find a very even distribution with virtually no top or bottom. And the last classification includes propositions 13 to 16, where the mean is low for three of the propositions, but higher for the fifteenth proposition. This illustration is really just another way of showing what has already been presented in the previous table. The visualization can possibly help with an enhanced visual appearance where the graph itself can contribute to the understanding and interpretation. 5. Discussion There are two areas that this paper has focused on. The first is about what should or could be included in the work with pedagogical communication of financial information. This has mainly focused on the theoretical review, which in turn formed the basis for the second part: how the pedagogical communication is perceived by CFO’s in practice in Swedish municipalities. This discussion will be organized according to these two areas, followed by conclusions. 5.1 The design of pedagogical communication, focusing on economic information After reviewing different theories and previous research, a number of words and concepts crystallized, which came to play a central role in the paper. These can, in a sense, summarize what pedagogical communication means in a financial context. In the simplest form, communication is about a message transferred from a transmitter to a receiver. In this study, the transmitter is the CFO, which formed the basis for the practical part. The recipient receives something that is communicated, which in this paper is financial information. In a way, this is the essence of pedagogical communication. The concepts that have been used to www.macrothink.org/ijafr www.macrothink.org/ijafr 176 International Journal of Accounting and Financial Reporting ISSN 2162-3082 2013, Vol. 3, No. 1 capture this transfer are partly about the structuring of information: if the information is communicated by numbers or text, if it is readily available, if it is presented with graphic illustrations and the language used. But the study also included the forms in which communication takes place, such as verbal communication as a complement to the written reports. Another important part of the pedagogical communication is that several theories deal with meaning and understanding, a meaning for the recipient. It has also become clear that the communication from a transmitter is both to an individual and to a group of receivers. According to the theories, the group, together, interprets the meaning, , enabling a common understanding where meaning is created. Again, the alignment of communication and meaning is at the center. As a final step in a planned sequence, the communication of financial information works as a basis for analysis and decision. These concepts and areas are a form of operationalization of the meaning of pedagogical communication of financial information. A somewhat more difficult question is how the pedagogical communication should be designed. The simplest answer would be that it depends on the organization. Another natural starting point would be to focus on the keywords identified in the theory section, which are summarized in the paragraph above. The design is probably more a matter of thinking through four different perspectives, e.g. operationalization of the pedagogic communication. A somewhat more difficult question is how the pedagogical communication should be designed. The simplest answer would be that it depends on the organization. Another natural starting point would be to focus on the keywords identified in the theory section, which are summarized in the paragraph above. The design is probably more a matter of thinking through four different perspectives, e.g. operationalization of the pedagogic communication. It has become clear that the report or the written communication is a natural and obvious base for the financial communication, which forms the first group. These are frequently used, and have in a way become synonymous with communication. However, one may ask if the sender reflects on how the report is received, and if the meaning is understood. It is also natural to talk about the financial information as something that is communicated verbally, often in different meetings and briefings. This is the second group. These become a natural context in which meanings and understanding are important. In a conversation, a two-way communication, it becomes clear if the recipient has understood what has been said. The core issue of the design is here more about the contexts in which financial information is presented, and how an oral presentation becomes a written supplement. In a third group, the content of the communication was in the center, particularly the concepts and illustrations. Here, the focus is on the transmitter’s purpose of the communication and the concepts used. A fourth group focuses on pedagogical communication where the recipient is not a person but a group. This is of course a natural starting point for many organizations, especially where work is not done individually, but in groups. Again, the meaning and understanding is at the center, which in a sense can be described as the outcome and purpose of communication. The concepts and the four groupings constitute a basis for how the pedagogical communication of financial information can be understood and developed. Based on the type of organization and the type of leader the CFO wants to be, there are various possible approaches and priorities. The common denominator is that the four areas can contribute to create a more pedagogical communication. The next part of the paper is about how pedagogical communication of financial information is done in practice, from a CFO perspective 177 www.macrothink.org/ijafr www.macrothink.org/ijafr 5.2 How the pedagogical communication works in Swedish municipalities 5.2 How the pedagogical communication works in Swedish municipalities The first impression is that CFO’s see themselves as very skilled at communicating financial information in a pedagogical way. If the Likert scale is divided into three parts, the answers 1, 2, 3 is a low group where you do not work with the area concerned in a particular proposition. The second part is in between with the answer 4, and the third part is answers 5, 6, 7, where a group agrees on what the proposition describes. There are several statements where an overwhelming majority of the 111 respondents think that they recognize themselves in the third part. 101 respondents regularly communicate financial information orally; 95 respondents want to use an accessible, easy-to-understand language,: 93 respondents communicate financial information both in writing and in figures; 93 respondents think it is important to communicate a meaning and not just numbers; 93 respondents clarify a meaning if the receiver has questions related to an oral presentation and 91 respondents answer that it is very common that the financial information underlies the analyzes and decisions. Within these six areas, an overwhelming majority feel at home. However, you are less able to adapt the structure of the reports, 53 respondents answered 5, 6 or 7; information is interpreted together, 60 respondents answered and it is not so certain that the receiver knows the meaning of the terms used, 69 respondents answered, to take three examples. These are potential areas for development. The results also show that in many cases there are natural, almost normal distribution, patterns among the responses. The respondents perceive that they are working with the areas this paper has identified as central to financial information. www.macrothink.org/ijafr International Journal of Accounting and Financial Reporting ISSN 2162-3082 2013, Vol. 3, No. 1 5.2 How the pedagogical communication works in Swedish municipalities International Journal of Accounting and Financial Reporting ISSN 2162-3082 2013, Vol. 3, No. 1 cal communication works in Swedish municipalities International Journal of Accounting and Financial Reporting ISSN 2162-3082 2013, Vol. 3, No. 1 6. Conclusions Several different conclusions about how pedagogical communication of financial information should be designed and how it works in practice could be recognized. Pedagogical communication is much more than just a report. It has become clear that the report itself is central and still very important, but it is even more important to communicate a meaning and understanding. CFO’s in Swedish municipalities actively work with most of the key areas within pedagogical communication, as defined and described in this study. Despite the high number of responses to many of the propositions, there are still areas in which development can take place, such as the group's ability to understand and interpret a report, e.g. create meaning, It is common to many of the propositions and responses gatheredthat it is the result of communication, the understanding and creation of meaning that is important. Communication is the agent, which generates a result. In this paper, the educational communication has focused on the importance of communicating a meaning and a purpose. This is theoretically based on Checkland and Holwells (1998) theory of organizational sensemaking and Weicks (2001) theories of sensemaking. Several of the propositions had this focus, and some propositions had that meaning even though it is not explicitly requested. A summary conclusion could possibly be that the respondents have the ambition to communicate a meaning and a purpose, while they are not always sure that the recipients of the information are able to interpret the information and understand its meaning. In the light of this reasoning, one can possibly see the financial www.macrothink.org/ijafr 178 2013, Vol. 3, No. 1 reports, the oral presentation, the content of the reports and the group as tools that will lead to understanding and meaning being communicated. reports, the oral presentation, the content of the reports and the group as tools that will lead to understanding and meaning being communicated. As the CFO’s formed the empirical material in this study, it would have been interesting to compare their perceptions with the perception of those who they communicate with. To compare the transmitter and receiver perceptions of financial information could provide a deeper understanding of pedagogical communication. It would also have been interesting to conduct interviews with some selected CFO’s in comparable municipalities to get a deeper understanding of how they work with communication in practice. There is much more that can be studied in the context of pedagogical communication, and ultimately communicative learning processes. Englund (2007) argues that it is a virtually unexplored area. Meaningful pedagogical communication processes is thus the future. References Abdullah Zaid, O., & Abraham, A. (1994). Communication skills in accounting education: perceptions of academics, employers and graduate accountants. Accounting Education, 3(3), 205-221. Alter, S. (2002). Information systems. NJ; Upper Saddle River: Prentice Hall. Andrews, J. D., & Koester, R. J. (1979). Communication difficulties as perceived by the accounting profession and professors of accounitng. Journal of Business Communication, 16(33). Andrews, J. D., & Koester, R. J. (1979). 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https://openalex.org/W4225886668	https://www.researchsquare.com/article/rs-1510160/latest.pdf	English	null	Development and validation of a scale for the tendency to exercise in response to mood, eating, and body image cues: The Reactive Exercise Scale (RES)	Research Square (Research Square)	2,022	cc-by	11,568	Development and validation of a scale for the tendency to exercise in response to mood, eating, and body image cues: The Reactive Exercise Scale (RES) Kyle De Young (  kyle.deyoung@uwyo.edu ) University of Wyoming https://orcid.org/0000-0002-6307-4029 P. Evelyna Kambanis University of Wyoming Angeline R. Bottera University of Wyoming Christopher J. Mancuso University of Wyoming Kyle De Young (  kyle.deyoung@uwyo.edu ) University of Wyoming https://orcid.org/0000-0002-6307-4029 P. Evelyna Kambanis University of Wyoming Angeline R. Bottera University of Wyoming Christopher J. Mancuso University of Wyoming Abstract Purpose: Earlier work on engaging in physical exercise when experiencing negative affect demonstrated robust associations with eating disorder (ED) behaviors and attitudes; however, measurement of the behavior was primitive. We report on the development of a self-report measure, the Reactive Exercise Scale (RES), that disentangles the tendency to engage in exercise in response to negative mood cues from the tendency to engage in exercise in response to eating and body image cues, which themselves may be associated with negative mood. The measure also assesses exercising in response to positive mood cues. Method: Exploratory factor analysis (FA) guided item and factor selection. Confirmatory FA in an independent sample supported a 3-factor solution – exercising in response to negative mood cues, eating and body image cues, and positive mood cues. Results: Results supported the construct validity of the RES subscales and indicated that exercising in response to negative mood cues may not uniquely relate to most aspects of ED psychopathology when accounting for eating and body image cues, which themselves are associated with negative mood. Conclusion: The RES allows for testing the unique relation of exercise to regulate negative emotion with ED constructs independent from the appearance-related motives for which exercise may also be used. Research Article Research Article Keywords: exercise, eating disorders, negative mood, positive mood, body image Posted Date: April 5th, 2022 DOI: https://doi.org/10.21203/rs.3.rs-1510160/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/26 Page 1/26 Page 1/26 What Is Already Known On This Subject? Exercise behavior is often disrupted in individuals with EDs and itself may be an important ED behavior. The aspects of exercise that explain its relation to ED features, and what differentiates exercise as it occurs within and outside EDs, remains an important topic of study. What This Study Adds? This study contributes a novel measure of the tendency to engage in exercise in response to specific cues: negative emotion, eating/body image, and positive emotion. Together, these subscales may aid in identifying the specific components of exercise that relate to ED features from those that may occur both within and outside of EDs. Introduction This work has the potential to support research on the emotion regulatory aspects of exercise and the associations of such exercise with ED psychopathology, clarifying the extent to which exercise itself may be a treatment target and how it might best be altered to support adaptive behavior. In fact, exercising in response to negative affect, which may reflect using exercise to regulate emotions, is both common and robustly associated with ED psychopathology. For instance, 58% of college students reported having ever engaged in exercise because they were experiencing negative mood, and these students were much more likely to endorse current ED thoughts and behaviors (De Young & Anderson, 2010). Distinguishing participants by whether they ever exercised in response to negative mood resulted in large effects on measures of ED behaviors and psychopathology, in some cases being the only unique predictor of these outcomes when included among existing exercise dimensions known to be related to ED constructs (e.g., obligatory exercise). Importantly, in the cited study, exercise in response to negative mood was assessed with the use of a single dichotomous item, which precluded thorough analysis of the relations between ED constructs and the dimension possibly underlying this dichotomous item. The goal of the studies we report on here was to develop a measure of this dimension in addition to other similar dimensions that might be important for clarifying the relations between the tendency to exercise in response to negative mood cues and ED constructs. This work has the potential to support research on the emotion regulatory aspects of exercise and the associations of such exercise with ED psychopathology, clarifying the extent to which exercise itself may be a treatment target and how it might best be altered to support adaptive behavior. To date, most concepts of exercise in association with ED psychopathology have focused on the extent to which individuals feel compelled or obligated to engage in the exercise, and a variety of terms have been used to describe these aspects of exercise (Meyer & Taranis, 2011). For instance, obligatory exercise involves experiencing negative emotion, such as feelings of guilt, depression, and anxiety, as a consequence of not exercising (Meyer et al., 2011). Exercising is understood to alleviate or prevent such negative emotional consequences among individuals who engage in obligatory exercise, indicating that it may also function to regulate emotion (Stice, 1994; Whiteside et al., 2007). Introduction Exercise has mood-regulating and stress-reducing effects (Cash et al., 1994; Thayer et al., 1994; Taranis et al., 2011). Specifically, exercise is associated with increases in positive affect and decreases in negative affect (Giacobbi et al., 2006; Bodin & Martinsen, 2004), including reducing anxiety (Asmundson et al., 2013; Schuch et al., 2016). Generally speaking, exercise interventions have beneficial physical and psychological effects in individuals with psychiatric disorders (Szuhany et al., 2015; Sachs & Buffone, 1984). Among individuals with eating disorders (EDs), although the primary motive of compulsive exercise is to control shape and weight (Dalle Grave et al., 2008), individuals often report using exercise to regulate mood (Meyer et al., 2011), and evidence supports pre- to post-exercise decreases in negative emotion in individuals with Page 2/26 Page 2/26 Page 2/26 EDs (Engel et al., 2013; Noetel et al., 2016). Thus, the mood regulating effects of exercise can be understood as one of its benefits; however, exercise behavior as it occurs among individuals with EDs has been the subject of scrutiny for its possible role as a problematic behavior targeted for reduction in treatment (e.g., Martenstyn et al., 2021; see also Cook & Leininger, 2017; Mathisen et al., 2021; Quesnel et al., 2018). In fact, exercising in response to negative affect, which may reflect using exercise to regulate emotions, is both common and robustly associated with ED psychopathology. For instance, 58% of college students reported having ever engaged in exercise because they were experiencing negative mood, and these students were much more likely to endorse current ED thoughts and behaviors (De Young & Anderson, 2010). Distinguishing participants by whether they ever exercised in response to negative mood resulted in large effects on measures of ED behaviors and psychopathology, in some cases being the only unique predictor of these outcomes when included among existing exercise dimensions known to be related to ED constructs (e.g., obligatory exercise). Importantly, in the cited study, exercise in response to negative mood was assessed with the use of a single dichotomous item, which precluded thorough analysis of the relations between ED constructs and the dimension possibly underlying this dichotomous item. The goal of the studies we report on here was to develop a measure of this dimension in addition to other similar dimensions that might be important for clarifying the relations between the tendency to exercise in response to negative mood cues and ED constructs. Introduction Obligatory exercise demonstrates a number of associations with ED variables that illustrate its importance as a way of conceptualizing problematic exercise in the context of EDs. Obligatory exercise is undertaken more frequently and intensely than non-obligatory exercise, and individuals who engage in obligatory exercise spend more time thinking about exercise (Steffen & Brehm, 1999). A systematic review concluded that obligatory exercise is associated with heightened eating and weight psychopathology (Meyer et al., 2011). In fact, obligatory exercise was reported by 20-80% of individuals with EDs (Shroff et al., 2006) and associated with poorer treatment outcome, illness chronicity, and severity of psychopathology (Dalle Grave et al., 2008; El Ghoch et al., 2013). Importantly, why individuals’ emotions are related to their exercise behavior has not been central to investigations of exercise in the context of ED psychopathology. Individuals may feel compelled to exercise, and feel guilty if they do not exercise, for a variety of reasons, including reasons related to their eating behaviors and body shape and weight, as well as reasons unrelated to eating, shape, and weight (e.g., athletic performance; Karr et al., 2013). However, because ED behaviors Page 3/26 Page 3/26 and body shape and weight concerns are themselves associated with negative emotion (e.g., Heron et al., 2014; Leahey et al., 2011), investigations of the relations between negative emotion, exercise, and ED psychopathology should account for eating behavior- and body image-driven exercise. In the absence of parsing these effects, evidence might indicate relations between negative emotion, exercise, and ED psychopathology that are better explained by the eating and body image exercise motives held by individuals with ED psychopathology who exercise, which themselves are associated with negative mood. Thus, the goal of the following studies was to develop a measure - the Reactive Exercise Scale (RES) - for assessing the dimension of negative mood cued exercise that also assesses eating cued and body image cued exercise so that the unique relations of negative mood cued exercise and ED psychopathology can be examined. In addition, we aimed to assess positive mood cued exercise to test whether the valence of mood leading to exercise is important to consider and to aid in establishing the construct validity of the scale. Procedure The local institutional review board approved this study. Undergraduate students from a Northern Plains university participated in an online study (hosted on Qualtrics) for credit toward course requirements that was advertised as a survey study of eating and exercise. After providing informed consent, participants completed a series of self-report measures. Introduction and body shape and weight concerns are themselves associated with negative emotion (e.g., Heron et al., 2014; Leahey et al., 2011), investigations of the relations between negative emotion, exercise, and ED psychopathology should account for eating behavior- and body image-driven exercise. In the absence of parsing these effects, evidence might indicate relations between negative emotion, exercise, and ED psychopathology that are better explained by the eating and body image exercise motives held by individuals with ED psychopathology who exercise, which themselves are associated with negative mood. Thus, the goal of the following studies was to develop a measure - the Reactive Exercise Scale (RES) - for assessing the dimension of negative mood cued exercise that also assesses eating cued and body image cued exercise so that the unique relations of negative mood cued exercise and ED psychopathology can be examined. In addition, we aimed to assess positive mood cued exercise to test whether the valence of mood leading to exercise is important to consider and to aid in establishing the construct validity of the scale. Study 1 In Study 1, we aimed to develop an initial item pool and conduct an exploratory factor analysis (EFA) to gather preliminary evidence of the convergent and discriminant validity of the RES factors in an undergraduate student sample. We wrote items intending to characterize tendencies to engage in exercise in response to certain distinct moods, thoughts, and perceptual cues. These included negative mood cued exercise, eating cued exercise, body image cued exercise, and positive mood cued exercise. Broadly, we hypothesized that: (a) negative mood cued exercise would be positively associated with measures of psychopathology and emotion regulation, but not with non-mood related aspects of exercise; (b) eating cued and body image cued exercise would be positively associated with ED psychopathology, but not with aspects of emotion regulation; and (c) positive mood cued exercise would be negatively associated with measures of psychopathology, and not associated with emotion regulation. Measures Reactive Exercise Scale (RES). The initial version of the RES contained 42 items rated on a visual analog scale, with endpoints anchored by “Not at all” and “Extremely”. The instructions asked participants to “Please read each of the following statements. Using the scale provided, select the point on the line to indicate how likely it is that you would exercise given the situation described in the statement.” Items are listed in Table 1. We wrote the initial RES items using a top-down approach to capture four domains that might cue exercise behavior. They were: (a) exercising in response to negative mood cues; (b) exercising in response to eating cues; (c) exercising in response to body image cues; and (d) exercising in response to positive mood cues. Difficulties in Emotion Regulation Scale (DERS). The DERS (Gratz & Roemer, 2004) is a 36-item self-report measure of dimensions of emotion regulation, measured on six facets: nonacceptance of emotional responses (α = .92); difficulty engaging in goal-directed behavior (α = .89); impulse control difficulties (α = .86); lack of emotional awareness (α = .84); limited access to emotion regulation strategies (α = .91); and lack of emotional clarity (α = .77), and a total score (α = .94). Each item is rated on a scale of 1 (Almost never) to 5 (Almost always), with higher scores indicative of greater difficulties in emotion regulation. Positive and Negative Affect Schedule (PANAS-X). The PANAS-X (Watson & Clark, 1999) contains measures of negative and positive affect in addition to several more specific facets. We included the two 10-item affect scales to measure positive affect (α = .89) and negative affect (α = .89) and used the “in general” timeframe in the instructions. We also included 13 additional items that allowed for the measurement of sadness (α = .90), guilt (α = .91), and fatigue (α = .88). Each item is rated on a 5-point scale ranging from 1 (Very slightly or not at all) to 5 (Extremely). Higher scores are indicative of more intense affect. International Personality Item Pool (IPIP). The 142 items from the IPIP (Goldberg et al., 2006) approximate Cloninger et al.’s (1994) Temperament and Character Inventory dimensions of novelty-seeking (α = .82), harm avoidance (α = .80), and reward dependence (α = .83) and Carver and White’s (1994) Behavioral Inhibition and Activation Scales. Participants Participants were 315 undergraduate students. Their mean (SD) age was 19.6 (3.0) years and 82.5% identified as female. Their mean (SD) body mass index (BMI) based on self-reported height and weight was 23.4 (4.0) kg/m2. The majority identified as non-Hispanic, White (91.1%), with fewer identifying as Black/African American (1.0%), Asian (2.9%), Native American (1.3%), Hispanic (0.6%), or other (1.3%; 1.9% did not provide information about their race/ethnicity). [Table 1 here] [Table 1 here] Page 4/26 Page 4/26 Results Sample size per RES item ranged from 269-296. The Kaiser-Meyer-Olkin measure of sampling adequacy was .959, above the recommended value of .6 (Costello & Osborne, 2005), and Bartlett’s test of sphericity was significant (c2 = 12359.51; df = 861; p < .001), indicating that the data were appropriate for an EFA. Examination of the scree plot, initial eigenvalues, and results of a parallel analysis all indicated that three factors should be extracted (Figure 1). Together, these three factors explained 67.09% of the inter-item variance. We retained five items (items 4, 8, 9, 14, and 22; Table 1) despite cross-loadings greater than 0.32 (Tabachnik & Fidell, 2013), because we judged their content to be central to the construct being measured or important for covering the breadth of the construct (i.e., they were critical for content validity). In total, we retained 25 of the 42 items. Measures The behavioral inhibition system (BIS) corresponds to motivation to avoid aversive outcomes, and the behavioral activation system (BAS) corresponds to motivation to approach goal-oriented outcomes. This measure contains four subscales, one for measuring the BIS dimension (α = .82), and three measures of the BAS dimension (Reward Responsiveness [BAS-RR; α = .67], Drive [BAS-D; α = .63], and Fun-Seeking [BAS-FS; α = .79]). IPIP items were self-assessed using a 1 (Very inaccurate) to 5 (Very accurate) rating scale. For all scales, higher scores indicate higher standing on the construct. Obligatory Exercise Questionnaire (OEQ). The OEQ (Pasman & Thomspon, 1988) is a 20-item self-report questionnaire consisting of statements about the extent to which an individual feels obligated or compelled to exercise. Statements are rated on a 4-point scale ranging from 1 (Never) to 4 (Always), with higher scores indicating more obligatory exercise. Cronbach’s alpha for the OEQ was .90. Reasons for Exercise Inventory (REI). The REI (Silberstein et al., 1988) is a 24-item self-report questionnaire that contains statements about motivation for exercise. Participants rate their motivations to engage in exercise on a 7-point scale ranging from 1 (Not at all important) to 7 (Extremely important). The subscales Page 5/26 Page 5/26 of reasons for exercise include weight control (α = .54); fitness (α = .91); health (α = .87); mood (α = .84); physical attractiveness (α = .87); enjoyment (α = .92); and body tone (α = .84). of reasons for exercise include weight control (α = .54); fitness (α = .91); health (α = .87); mood (α = .84); physical attractiveness (α = .87); enjoyment (α = .92); and body tone (α = .84). Statistical Analyses We conducted an EFA using principal axis factoring with varimax rotation in SPSS (IBM, 2019) to maximize variance in factor loadings and aid in achieving an interpretable solution in this early stage despite the likelihood that factors would be correlated (e.g., Kim & Meuller, 1978) to identify dimensions present in the RES. Pairwise exclusion was used for missing values. We visually examined the scree plot (Figure 1) and performed a parallel analysis (Horn, 1965) to determine the optimal number of factors to extract. Items that loaded above 0.60 with cross-loadings below 0.32 were automatically retained (Costello & Osborne, 2005; Tabachnick & Fiddell, 2013). We used partial correlations – controlling for RES subscales, sex, and BMI –to assess convergent and discriminant validity, using p < .05 as the statistical significance threshold to maximize detected relationships. [Figure 1 here] [Table 2 here] In Table 2, we provide means, standard deviations, bivariate Pearson correlations, and internal consistencies for the three extracted factors. The first extracted factor is represented by 12 items and indicates the tendency to engage in exercise in response to negative mood cues. The second extracted factor is represented by 10 items and indicates the tendency to engage in exercise in response to eating and body image cues. The third extracted factor is represented by three items and indicates the tendency to engage in exercise in response to positive mood cues. Because the eating and body image cues resided on the same factor, we also examined a four-factor solution. However, extracting a fourth factor did not lead the eating items to separate from the body image items. Instead, there was no clearly discernible pattern to the item-factor configuration, further indicating that the three-factor solution fit better. [Table 3 here] Page 6/26 In Table 3, we provide preliminary evidence of convergent and discriminant validity. The negative mood factor was positively associated with the OEQ total score and the REI mood subscale but negatively associated with the REI weight control subscale. It was also positively associated with the DERS impulse and total scores and PANAS sadness facet. The negative mood factor was not associated with behavioral activation, positive affect, novelty seeking, reward dependence, or fitness and health. The eating and body image factor was positively associated with REI weight control, BIS/BAS anxiety, PANAS negative affect and guilt facet, and IPIP novelty seeking. It was not associated with behavioral activation, positive affect, novelty seeking, reward dependence, and REI fitness and health motives. Finally, the positive mood factor was positively associated with OEQ obligatory exercise and was the only factor associated with the REI fitness and health subscales. The positive mood factor was also positively associated with PANAS positive affect and BAS drive and negatively associated with DERS impulse and total scores; PANAS negative affect guilt, and sadness; and IPIP harm avoidance. With regard to discriminant validity, the positive mood factor was not associated with behavioral inhibition, novelty seeking, reward dependence, mood, or weight control. Procedure The local institutional review board approved this study. Undergraduate students from a Northern Plains university participated in an online study (hosted on Qualtrics) for credit toward course requirements advertised as a survey study about exercise, eating behavior, and mental health. Discussion The results of Study 1 preliminarily indicate that a 25-item version of the RES is composed of three dimensions representing the tendencies to exercise in response to negative mood cues, eating and body image cues, and positive mood cues. These scales appear to have excellent internal consistency reliability, indicating low idiosyncratic error. Despite using an orthogonal rotation, factors were moderately related, which is common (Nunnally & Bernstein, 1994). Study 2 In Study 2, we tested the fit of the 3-factor solution that resulted from Study 1 in an independent undergraduate student sample. We included measures of ED psychopathology and other clinical syndromes (i.e., depression, self-injury, anxiety, and alcohol use). We hypothesized that, when controlling for sex, BMI, and the two other RES subscales (due to their moderate inter-correlations), (a) negative mood-cued exercise would be positively associated with measures of psychopathology but not ED-specific psychopathology; (b) eating and body image-cued exercise would be positively associated with ED psychopathology; and (c) positive mood-cued exercise would be negatively associated with psychopathology. Participants Page 7/26 Measures Eating Disorder Examination – Questionnaire (EDE-Q). The EDE-Q (Fairburn & Beglin, 1994) is a 28-item self-report questionnaire that assesses the frequency of eating behaviors over the prior 28-days and provides indications of eating psychopathology on four subscales (Dietary Restraint [α = .87], Eating Concern [α = .88], Shape Concern [α = .94], and Weight Concern [α = .90]), which are averaged to form a Global Score (α = .96). Eating Pathology Symptoms Inventory (ESPI). The EPSI (Forbush et al., 2013) is a 45-item self-report questionnaire assessing eight subscales: Body Dissatisfaction (α = .91); Binge Eating (α = .86); Cognitive Restraint (α = .75); Purging (α = .86); Restricting (α = .85); Excessive Exercise (α = .91); Negative Attitudes Toward Obesity (α = .88); and Muscle Building (α = .82). Participants respond to each item using a 5-point scale from 0 (Never) to 4 (Often) describing how much each item characterizes their experiences, with higher scores indicating higher standing on the dimensions. Clinical Impairment Assessment (CIA). The CIA (Bohn et al., 2008) is a 16-item self-report measure assessing psychosocial impairment due to ED symptoms over the prior 28 days. Participants rate the extent to which eating problems and weight and shape concerns have caused them impairment in various aspects of their life, with higher scores indicative of greater impairment. Cronbach’s alpha was .96. Multidimensional Body-Self Relations Questionnaire (MBSRQ). The MBSRQ (Cash, 2000) is a 69-item self- report questionnaire that assesses body image disturbance on 10 subscales. Participants rate the extent to which they agree with statements about their appearance from 1 (Definitely disagree) to 5 (Definitely agree). The subscales are Appearance Evaluation (α = .91), Appearance Orientation (α = .84), Fitness Evaluation (α = .81); Fitness Orientation (α = .90); Health Evaluation (α = .70); Health Orientation (α = .76); Illness Orientation (α = .74); and the Body Area Satisfaction Scale (BASS; α = .87). Center for Epidemiologic Studies Depression Scale-Revised (CESD-R). The CESD-R (Eaton et al., 2004) is a 20-item self-report measure of depression, based on the fourth edition of the Diagnostic and Statistical Manual of Mental Disorders (APA, 2000) criteria. Participants respond to questions asking about the frequency of symptoms of depression they have experienced over the prior week, ranging from 0 (Not at all) to 4 (A lot). Scores on the CESD-R range from 0-60, with higher scores indicating greater presence of depressive symptoms. Cronbach’s alpha was .95. Page 7/26 Participants were 300 undergraduates. Their mean (SD) age was 19.8 (1.9) years, 73.4% reported female sex, and 72.9% reported identifying as women. Their mean (SD) BMI based on self-reported height and weight was 24.4 (4.6) kg/m2. The majority identified as non-Hispanic, White (84.9%), with fewer identified as Hispanic (9.9%), Asian (2.3%), Black (0.7%), or other/mixed (2.3%). Participants were 300 undergraduates. Their mean (SD) age was 19.8 (1.9) years, 73.4% reported female sex, and 72.9% reported identifying as women. Their mean (SD) BMI based on self-reported height and weight was 24.4 (4.6) kg/m2. The majority identified as non-Hispanic, White (84.9%), with fewer identified as Hispanic (9.9%), Asian (2.3%), Black (0.7%), or other/mixed (2.3%). Participants were 300 undergraduates. Their mean (SD) age was 19.8 (1.9) years, 73.4% reported female sex, and 72.9% reported identifying as women. Their mean (SD) BMI based on self-reported height and weight was 24.4 (4.6) kg/m2. The majority identified as non-Hispanic, White (84.9%), with fewer identified as Hispanic (9.9%), Asian (2.3%), Black (0.7%), or other/mixed (2.3%). Participants were 300 undergraduates. Their mean (SD) age was 19.8 (1.9) years, 73.4% reported female sex, and 72.9% reported identifying as women. Their mean (SD) BMI based on self-reported height and weight was 24.4 (4.6) kg/m2. The majority identified as non-Hispanic, White (84.9%), with fewer identified as Hispanic (9.9%), Asian (2.3%), Black (0.7%), or other/mixed (2.3%). Measures Alcohol Use Disorders Identification Test (AUDIT). The AUDIT (Saunders et al., 1993) is a 10-item measure assessing alcohol consumption, drinking behaviors, and alcohol-related problems. Items measure typical Page 8/26 Page 8/26 Page 8/26 frequency, typical quantity per occasion, and heavy episodic drinking (six or more drinks per occasion) frequency. Participants respond to statements indicating the frequency that they have engaged in each statement, with response options coded from 0-4 and higher scores indicating more problems with alcohol. Cronbach’s alpha was .81. State-Trait Inventory for Cognitive and Somatic Anxiety (STICSA). The STICSA (Ree et al., 2008) is a self- report measure assessing cognitive and somatic symptoms of anxiety as pertaining to one’s current feelings of anxiety (state) and one’s general, overall feelings of anxiety (trait). Participants rate each item on a 4-point scale, ranging from 1 (Not at all) to 4 (Very much so). Cronbach’s alpha was .94 for the total trait measure, which is what we used here. Brief Experiential Avoidance Questionnaire (BEAQ). The BEAQ (Gámez et al., 2014) is a brief measure of the Multidimensional Experiential Avoidance Questionnaire (MEAQ). It is a 15-item self-report questionnaire assessing experiential avoidance. Each item is scored on a 6-point scale ranging from 1 (Strongly disagree) to 6 (Strongly agree). Higher scores are indicative of greater levels of experiential avoidance. Cronbach’s alpha was .87. Inventory of Statements about Self-Injury (ISAS). The ISAS (Klonsky & Glenn, 2009) assesses lifetime frequency of non-suicidal self-injurious (NSSI) behaviors performed “intentionally (i.e., on purpose) and without suicidal intent (i.e., not for suicidal reasons).” The behaviors assessed include banging/hitting self, biting, burning, carving, cutting, wound picking, needle-sticking, pinching, hair-pulling, rubbing skin against rough surfaces, severe scratching, and swallowing chemicals. Participants were asked to estimate the number of times in their lives they have performed each behavior, which we dichotomized as ever present versus absent. Statistical Analyses We conducted a confirmatory factor analysis (CFA) using Mplus (Version 8.3; Muthén & Muthén, 2017) to test the fit of the 3-factor solution using the MLR estimator. We assessed fit using the root mean square error of approximation (RMSEA; < .08 is fair; Brown & Cudeck, 1993), comparative fit index (CFI; > 0.95 is excellent; Hu & Bentler, 1999), Tucker-Lewis index (TLI; > 0.90 is acceptable; Bentler & Bonett, 1980), and the standardized root mean square residual (SRMR; < 0.06 is ideal; Hu & Bentler, 1999). We calculated partial correlations between the RES scales and other study measures while controlling for the other RES scales and participant sex and BMI. We conducted independent samples t-tests to examine whether scores on each subscale differed by sex. Finally, we conducted a multiple linear regression to account for EDE-Q Global Score, a broad measure of ED psychopathology, using the RES scales, sex, and BMI to examine which, if any, RES scales were uniquely associated with this important indicator of ED psychopathology while accounting for their shared variance. Results Page 9/26 After freeing the covariances of similarly worded items, the solution fit well (c2(263) = 485.33, p < .001; RMSEA = .053 [95%CI: .046, .061]; CFI = 0.95; TLI = 0.94; SRMR = 0.05). The factors indicate the tendency to exercise in response to negative mood cues, in response to eating and body image cues, and in response to positive mood cues (Figure 2). After freeing the covariances of similarly worded items, the solution fit well (c2(263) = 485.33, p < .001; The Tendency to Exercise in Response to Negative Mood Cues Exercising in response to negative mood cues was positively associated with the EDE-Q item assessing driven exercise, the EPSI excessive exercise and muscle building subscales, and the MBSRQ fitness and health orientation subscales. It was not associated with ED psychopathology subscales that do not assess exercise (e.g., dietary restraint, shape concern, restriction, impairment). The negative mood subscale was also not associated with any non-ED psychopathology measures. [Table 4 here] [Table 4 here] [Figure 2 here] Male participants scored higher on the negative mood cue subscale (44.62 ± 25.61) than female participants (34.51 ± 27.01; t(294) = -2.90, p = .004, d = 0.38), and they scored higher on the positive mood cue subscale (65.03 ± 28.43) than female participants (56.83 ± 30.02; t(294) = -2.12, p = .035, d = 0.28). In contrast, female participants scored higher on the eating and body image cued subscale (53.60 ± 26.95) than male participants (44.20 ± 25.12; t(294) = 2.71, p = .007, d = -0.36). Results of correlations between RES scales and other measures are displayed in Tables 3 and 4. The results of the multiple linear regression on the EDE-Q Global Score using all three RES scales indicated that, while controlling for sex and BMI, only the exercising in response to eating and body image cues factor was uniquely associated with EDE-Q Global Score (B = 0.51, t = 7.83, p < .001). [Table 3 here] [Table 3 here] [Table 3 here] [Table 4 here] The Tendency to Exercise in Response to Eating and Body Image Cues Exercising in response to eating and body image cues was associated with higher scores on the EDE-Q dietary restraint and eating concern subscales; EPSI restriction, cognitive restraint, and purging subscales; the presence of objectively large binge-eating episodes and purging as measured by the EDE-Q; and ED- related impairment as measured by the CIA. It was also positively associated with concerns regarding shape and weight (EDE-Q Shape Concern and Weight Concern) and negative attitudes toward obesity (EPSI Negative Attitudes toward Obesity). Higher scores on this dimension were positively associated with body dissatisfaction (EPSI Body Dissatisfaction) and appearance orientation (MBSRQ Appearance Orientation) and negatively associated with body image satisfaction (MBSRQ Appearance Evaluation, BASS) and health evaluation (MBSRQ Health Evaluation). Exercising in response to eating and body image cues was associated with greater endorsement of driven and excessive exercise (EDE-Q Driven Exercise and EPSI Excessive Exercise) and worse depression (CESD-R) and experiential avoidance (BEAQ). Finally, this dimension was not associated with binge eating measured by the EPSI, objectively large overeating Page 10/26 Page 10/26 episodes as measured by the EDE-Q, muscle building, fitness evaluation and orientation, health orientation, illness orientation, or depression, presence of self-injury, anxiety, and alcohol use. The Tendency to Exercise in Response to Positive Mood Cues No significant correlations were found between the tendency to exercise in response to positive mood cues and ED psychopathology or measures of other psychopathology. Exercising in response to positive mood cues was negatively associated with EPSI body dissatisfaction and positively associated with MBSRQ body satisfaction and appearance evaluation. It was also positively associated with EPSI excessive exercise and MBSRQ health and fitness evaluation and orientation. Further, the positive correlation between the RES Positive Mood subscale and MBSRQ Illness Orientation subscale suggests that exercising in response to positive mood is associated with increased alertness about, or reactivity to, being sick. An additional negative association was found between RES Positive Mood and BEAQ experiential avoidance. Exercising in response to positive mood cues was not associated with anxiety, depression, alcohol use problems, self- injury, age, or BMI. Study 2 Discussion The results of Study 2 provided support for the three-factor structure identified in Study 1, albeit with minor modifications to address correlated errors between similarly worded items. Convergent and discriminant evidence indicates that the negative mood cue subscale measures the tendency to engage in exercise in response to negative mood cues that are not specific to ED psychopathology, but are associated with other aspects of mental health, generally involving low mood and mood regulation. However, the negative mood factor did not demonstrate many associations with psychopathology, contrary to our prediction. Conversely, the eating and body image cues subscale appears to measure the tendency to engage in exercise that is related to ED psychopathology. Finally, the positive mood cue subscale appears to measure exercise that is generally associated with favorable mental health and not associated with psychopathology, ED or otherwise. Importantly, all three subscales demonstrate unique relations with the EPSI Excessive Exercise scale, and both the negative mood cue and the eating and body image cue subscales had unique relations with the EDE-Q question assessing driven exercise. Differences by sex indicated that male participants report stronger tendencies to exercise in response to mood cues but weaker tendencies to exercise in response to eating and body image cues than female participants, the latter of which is consistent with sex differences commonly observed on dimensions of ED psychopathology. Discussion Overall, these results support the construct validity of the RES subscales and indicate that exercising in response to negative mood cues may not uniquely relate to ED psychopathology, or perhaps even other forms of psychopathology, when accounting for eating and body image cues, which themselves may be associated with negative mood. Exercising in response to negative mood cues was positively associated with difficulties with emotion regulation, sadness, and fitness and health orientations. It was also associated with driven exercise, excessive exercise, and muscle building. It was not associated with problems with alcohol, depression, self- injury, or anxiety, which was counter to our expectations. Thus, while the RES appears to capture the tendency to exercise in the presence of negative mood cues, and this tendency is stronger the more one has difficulty regulating emotion or experiences sadness, this tendency does not relate to frank Notably, results in both studies point to the RES subscales being uniquely related to existing measures of obligatory exercise (i.e., the OEQ, EPSI Excessive Exercise, and less so with EDE-Q driven exercise). While this could be interpreted as the RES scales demonstrating a lack of discriminant validity with obligatory exercise, we think these findings reflect a strength of the RES compared to many existing measures of exercise quality employed in the ED literature. Specifically, individuals may feel compelled to exercise, and guilty if they do not exercise, for a variety of reasons. Certainly, some of those reasons are part and parcel of ED psychopathology and rightly point to such exercise as problematic in its own right. However, individuals who adopt forms of exercise as a valued, generally health-promoting, non-professional activity may set goals for their progress and improvement to which they commit themselves. Much like any other valued activity to which one commits, individuals may prioritize the activity, look forward to engaging in it, and be disappointed in general and perhaps even with themselves (i.e., feel guilty) when they fail to meet their own expectations. That disappointment may provide future motivation to compel their continued valued engagement. In fact, guilt may be understood as an apdaptive emotional experience when the opportunity for reparative actions are available (Tangney et al., 1992; Dahl et al., 2003). Discussion Earlier work on the tendency to engage in exercise when experiencing negative mood demonstrated that it was robustly associated with ED behaviors and attitudes (De Young & Anderson, 2010). However, given the benefits of exercise for mood and the common overlap in motives for exercise in EDs, the extent to which exercise in response to negative mood may be pathological is unclear. Thus, we aimed to devise a measure, the RES, to disentangle exercising in response to negative mood cues from exercising in response to eating and body image cues, which themselves may be associated with negative mood. We also included a Page 11/26 dimension of exercising in response to positive mood cues, in part to aid in establishing discriminative validity (i.e., that exercising in response to negative mood cues is distinct from exercising in response to positive mood cues). We utilized EFA to guide our item and factor selection and CFA in an independent sample to test the fit of our three-factor solution: exercising in response to negative mood cues, exercising in response to eating and body image cues, and exercising in response to positive mood cues. We had initially expected eating and body image cues to represent distinct dimensions, but this was not supported. Exercising in response to negative mood cues was positively associated with difficulties with emotion regulation, sadness, and fitness and health orientations. It was also associated with driven exercise, excessive exercise, and muscle building. It was not associated with problems with alcohol, depression, self- injury, or anxiety, which was counter to our expectations. Thus, while the RES appears to capture the tendency to exercise in the presence of negative mood cues, and this tendency is stronger the more one has difficulty regulating emotion or experiences sadness, this tendency does not relate to frank psychopathology. Consistent with our hypotheses, exercising in response to eating and body image cues was positively associated with nearly every ED-related variable we included and other measures of psychopathology, and was negatively associated with health evaluation. As hypothesized, exercising in response to positive mood cues was negatively associated with measures of psychopathology and positively associated with health, fitness, and appearance evaluation. Strengths and Limits We tested the validity of the RES using a homogenous sample in terms of sex, age, race, and ethnicity, limiting the generalizability of our findings to more diverse samples. Our use of undergraduate students in both RES studies also limits the generalizability of our findings to other populations. We have not yet tested the properties of the RES in a sample of individuals with EDs, so caution should be exercised when generalizing to that population. Our aim in developing the RES was to first ascertain reactive exercise in an undergraduate sample with a range of ED psychopathology prior to examining them in a clinical sample, in which restriction of range on variables of interest is likely. Future research should aim to examine the RES in clinical samples of individuals with EDs as well as individuals with other forms of psychopathology (e.g., depressive disorders). We did not examine the RES’ test-retest reliability or stability. Future research should examine its test-retest reliability over a multi-week (e.g., 4-week) interval, as the constructs assessed by the RES are theorized to be stable over such a period. Examining how RES subscale scores change over longer periods (e.g., from adolescence to adulthood) is an important avenue for future research. Finally, participants may not encounter visual analogue scales, such as the one used for the RES, as commonly as 4-7-point ordinal response scales; however, the ubiquitous use of online survey technology has led to the easy programming and scoring of these scales to address shortcomings of ordinal response scales (e.g., the tendency of participants to avoid extreme options on typical ordinal scales; Bishop & Herron, 2015). We encourage interested readers to contact us about how to program this scale in Qualtrics, as the use of a visual analogue scale makes paper and pencil scoring of this measure labor-intensive. Strengths of these studies include the use of two large, independent samples to establish the factor structure of the RES in separate exploratory and confirmatory steps, along with the inclusion of a large variety of variables to test convergent and discriminant relations. We tested the validity of the RES using a homogenous sample in terms of sex, age, race, and ethnicity, limiting the generalizability of our findings to more diverse samples. Our use of undergraduate students in both RES studies also limits the generalizability of our findings to other populations. Strengths and Limits We have not yet tested the properties of the RES in a sample of individuals with EDs, so caution should be exercised when generalizing to that population. Our aim in developing the RES was to first ascertain reactive exercise in an undergraduate sample with a range of ED psychopathology prior to examining them in a clinical sample, in which restriction of range on variables of interest is likely. Future research should aim to examine the RES in clinical samples of individuals with EDs as well as individuals with other forms of psychopathology (e.g., depressive disorders). We did not examine the RES’ test-retest reliability or stability. Future research should examine its test-retest reliability over a multi-week (e.g., 4-week) interval, as the constructs assessed by the RES are theorized to be stable over such a period. Examining how RES subscale scores change over longer periods (e.g., from adolescence to adulthood) is an important avenue for future research. Finally, participants may not encounter visual analogue scales, such as the one used for the RES, as commonly as 4-7-point ordinal response scales; however, the ubiquitous use of online survey technology has led to the easy programming and scoring of these scales to address shortcomings of ordinal response scales (e.g., the tendency of participants to avoid extreme options on typical ordinal scales; Bishop & Herron, 2015). We encourage interested readers to contact us about how to program this scale in Qualtrics, as the use of a visual analogue scale makes paper and pencil scoring of this measure labor-intensive. Strengths of these studies include the use of two large, independent samples to establish the factor structure of the RES in separate exploratory and confirmatory steps, along with the inclusion of a large variety of variables to test convergent and discriminant relations. Discussion Thus, whether such a pattern of exercise is problematic requires a detailed understanding of individuals’ values and other commitments and the extent to which their pursuit of exercise-related goals interferes with them; however, the findings of these studies with the RES seem to make clear that it is specifically exercising in response to eating and body image cues that uniquely relates to most aspects of ED psychopathology. This important Notably, results in both studies point to the RES subscales being uniquely related to existing measures of obligatory exercise (i.e., the OEQ, EPSI Excessive Exercise, and less so with EDE-Q driven exercise). While this could be interpreted as the RES scales demonstrating a lack of discriminant validity with obligatory exercise, we think these findings reflect a strength of the RES compared to many existing measures of exercise quality employed in the ED literature. Specifically, individuals may feel compelled to exercise, and guilty if they do not exercise, for a variety of reasons. Certainly, some of those reasons are part and parcel of ED psychopathology and rightly point to such exercise as problematic in its own right. However, Page 12/26 finding can help contextualize work focusing on understanding the emotion-regulation functions of exercise in EDs (Kolar & Gorrell, 2020). The RES appears to be a promising new tool for testing relations between exercise and ED psychopathology and how exercise cued by mood is implicated in these disorders. Indeed, while the ED literature presently contains a variety of measures for exercise (Harris et al., 2020), the results of our studies appear to indicate that the RES approaches characterizing the quality of exercise behavior uniquely from existing measures, and this may be advantageous for future work in this area. 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After a friend or loved one has let me down .778 .240 .243 21. When I feel guilty about something I’ve done or failed to do .775 .288 .240 18. After I’ve gotten into an argument .744 .178 .307 19. When I’m feeling like my future is uncertain .731 .182 .283 16. When I’m having a bad day .725 .226 .283 10. After a friend or loved one treated me badly .696 .260 .178 After I’ve disappointed myself .665 .394 .187 After not living up to my expectations .659 .453 .239 3. When I can’t make something perfect .653 .278 .269 2. When I’m feeling like I’m at my wit’s end .642 .306 .217 When I’m feeling anxious about how something will turn out .639 .240 .323 When I’m feeling like there’s nothing I can do to make things better .625 .340 .228 After I’ve been the butt of a joke .615 .433 .128 When I’m feeling worthless .554 .420 .242 17. After finding out that I’ve gained weight .229 .800 .247 12. After noticing that my clothes are too tight .232 .784 .177 7. After I’ve compared myself to someone in better shape than me .231 .774 .141 11. After someone has made a negative comment about the way I look .312 .746 .059 22. After realizing that I’m out of shape .239 .738 .364 8. After eating dessert .368 .737 .186 14. After I’ve eaten more than I had planned .339 .734 .197 6. After eating a large meal .261 .733 .140 Initial RES Items Factor 1 2 3 23. After I’ve let someone down .826 .255 .199 20. When I’m feeling like I’m a bad person .792 .292 .253 24. When I’m feeling depressed .789 .194 .214 25. When I’m feeling sad .787 .200 .242 13. After a friend or loved one has let me down .778 .240 .243 21. When I feel guilty about something I’ve done or failed to do .775 .288 .240 18. After I’ve gotten into an argument .744 .178 .307 19. When I’m feeling like my future is uncertain .731 .182 .283 16. Table 1 Exploratory factor analysis rotated factor matrix When I’m having a bad day .725 .226 .283 10. After a friend or loved one treated me badly .696 .260 .178 After I’ve disappointed myself .665 .394 .187 After not living up to my expectations .659 .453 .239 3. When I can’t make something perfect .653 .278 .269 2. When I’m feeling like I’m at my wit’s end .642 .306 .217 When I’m feeling anxious about how something will turn out .639 .240 .323 When I’m feeling like there’s nothing I can do to make things better .625 .340 .228 After I’ve been the butt of a joke .615 .433 .128 When I’m feeling worthless .554 .420 .242 17. After finding out that I’ve gained weight .229 .800 .247 12. After noticing that my clothes are too tight .232 .784 .177 7. After I’ve compared myself to someone in better shape than me .231 .774 .141 11. After someone has made a negative comment about the way I look .312 .746 .059 22. After realizing that I’m out of shape .239 .738 .364 8. After eating dessert .368 .737 .186 14. After I’ve eaten more than I had planned .339 .734 .197 6. After eating a large meal .261 .733 .140 After eating sweets .406 .697 .169 When I will feel guilty if I don’t exercise .305 .679 .401 15. When I’m planning on wearing a swimsuit .170 .670 .304 9. When I’m in a good mood .136 .375 .804 After I’ve gotten some good news .354 .133 .803 After accomplishing a goal that I set for myself .339 .213 .777 When I’m having a good day .076 .399 .765 After receiving a good grade in school or positive feedback at work .394 .095 .729 After I’ve accomplished something that was important to me .481 .104 .727 When I feel like I have complete control over my future .326 .240 .693 5. When I’m feeling hopeful about the future .172 .314 .688 4. After a good night’s rest .173 .379 .682 After having a good time with family or a friend .383 .162 .682 When I’m in the mood to celebrate .473 .099 .602 Note. Bolded numbers indicate highest factor loading for each item. Numbers on the left identify retained items and their order in the final measure and correspond to item numbers in Figure 2. Table 3 Unique relations between final Reactive Exercise Scale factors and other variables while controlling for sex and BMI in Study 1 Table 1 Exploratory factor analysis rotated factor matrix Factor 1 = negative mood cued exercise; Factor 2 = eating and body image cued exercise; Factor 3 = positive mood cued exercise. Table 2Des criptive statistics, Pearson correlation,s and internal consistencies of the Reactive Exercise Scale factors Study 1 Study 2 Pearson r Pearson r RES Subscale M(SD) α Eating/Body Image Positive Mood M(SD) α Eating/Body Image Positive Mood Negative Mood 39.64 (27.97) .961 .588 .483 37.29 (26.94) .948 .602 .545 Eating/Body Image 56.35 (27.24) .951 .584 51.01 (26.73) .932 .505 Positive Mood 61.46 (27.51) .903 58.98 (29.75) .883 Note. M = mean; SD = standard deviation; α = Cronbach’s alpha; All correlations are statistically significant at p < .001 (two-tailed). Table 2Des criptive statistics, Pearson correlation,s and internal consistencie Table 2Des criptive statistics, Pearson correlation,s and internal consistencies of the Reactive Exercise Scale factors Note. M = mean; SD = standard deviation; α = Cronbach’s alpha; All correlations are statistically significant at p < .001 (two-tailed). Note. M = mean; SD = standard deviation; α = Cronbach’s alpha; All correlations are statistically significant at p < .001 (two-tailed). Page 19/26 Page 19/26 Table 3 Unique relations between final Reactive Exercise Scale factors and other variables while controlling for sex and BMI in Study 1 Table 3 Unique relations between final Reactive Exercise Scale factors and other variables while controlling for sex and BMI in Study 1 Page 20/26 Page 21/26 Negative Mood Eating/Body Image Positive Mood IPIP BIS/BAS Anxiety Reward Response Drive Fun Seeking Goals -.060 -.044 -.025 .045 .038 .199*** .112 -.004 .111 -.058 -.060 .093 .146* .009 -.058 DERS Goals Impulse Non-Acceptance Awareness Strategies Clarity Total .038 .166** .126* .138* .138* .223* .197 .185** .052 .137* -.070 .109 -.009 .108 -.058 -.141* -.012 -.076 -.158* -.059 -.131* PANAS Positive Affect Negative Affect Guilt Sadness Fatigue -.023 .088 .114 .122* -.064 .065 .151* .229* .075 .102 .176 -.123* -.171** -.150* -.046 IPIP Harm Avoidance Worry/Pessimism Fear of Uncertainty Shyness Fatigue & Asthenia .021 .165 .006 .072 .203* -.022 -.041 .126* -.161 .036 -.112 -.211* IPIP Novelty Seeking Variety Seeking Recklessness Extravagance -.018 .023 .064 .105 .142* .000 .075 -.097 -.077 Negative Mood Eating/Body Image Positive Mood Negative Mood Eating/Body Image Negative Mood IPIP BIS/BAS IPIP BIS/BAS IPIP BIS/BAS Rebelliousness .000 .081 -.077 IPIP Reward Dependence Sentimentality Warm Communication Attachment Dependence .086 .081 .100 -.007 .041 .060 .015 .057 .014 .044 -.075 .064 OEQ Total .217*** .132* .196 REI Weight Control Fitness Mood Health -.252* -.038 .124* -.078 .403* .038 -.006 .070 .015 .259* .121 .225* Note. Values are partial correlations, controlling for sex, BMI, and the other RES scales themselves. BIS/BAS = Behavioral Inhibition Scale/Behavioral Activation Scale; DERS = Difficulties in Emotion Regulation Scale; PANAS = Positive and Negative Affect Scales; IPIP = International Personality Item Pool; OEQ = Obligatory Exercise Questionnaire; REI = Reasons for Exercise Inventory. p < .001; * p <.01; * p <.05. Table 4 Unique relations between final Reactive Exercise Scale factors and other variables while controlling .000 .081 -.077 -.077 .081 Rebelliousness Note. Values are partial correlations, controlling for sex, BMI, and the other RES scales themselves. BIS/BAS = Behavioral Inhibition Scale/Behavioral Activation Scale; DERS = Difficulties in Emotion Regulation Scale; PANAS = Positive and Negative Affect Scales; IPIP = International Personality Item Pool; OEQ = Obligatory Exercise Questionnaire; REI = Reasons for Exercise Inventory. *p < .001; p <.01; * p <.05. Table 4 Unique relations between final Reactive Exercise Scale factors and other variables while controlling for sex and BMI in Study 2 Page 22/26 Page 22/26 Negative Mood Eating/Body Image Positive Mood Age -.011 -.071 .056 BMI2 -.159** .031 .059 EDE-Q Dietary Restraint Eating Concern Weight Concern Shape Concern Driven Exercise Presence of OEs1 Presence of OBEs1 Presence of Purging1 .064 -.016 -.029 -.060 .134* .997 1.000 .994 .341* .279* .393* .438* .185 .997 1.020 1.035 .030 -.065 -.080 -.108 .084 .185 .992 .095 CIA Total Score .012 .310* -.056 EPSI Binge Eating Cognitive Restraint Purging Restriction Body Dissatisfaction Negative Attitudes Toward Obesity Excessive Exercise Muscle Building .000 .080 .041 .029 .012 -.004 .213* .127* .016 .348* .224* .151* .310*** .130* .160* .064 -.039 .158 -.082 -.071 -.056 .044 .260*** .096 MBSRQ Appearance Evaluation Appearance Orientation Fitness Evaluation Fitness Orientation Health Evaluation Health Orientation .077 -.110 .076 .153* .096 .149* -.224* .384* -.006 .015 -.133* .054 .158 -.019 .200* .346* .161 .238* MBSRQ Body Area Satisfaction Scale -.022 -.270* .214*** CESD-R .051 .145* -.079 AUDIT .036 .067 .026 STICSA .003 .020 -.008 BEAQ -.052 .163** -.118* Presence of Self-Injury1 1.003 1.002 .997 Note. Values are partial correlations, except where otherwise indicated, controlling for sex, BMI, and the other RES scales themselves. BMI = Body Mass Index; EDE-Q = Eating Disorder Examination – Questionnaire; OE = Objectively large overeating episodes; OBE = Objectively large binge eating episodes; CIA = Clinical Impairment Assessment; EPSI = Eating Pathology Symptom Inventory; MBSRQ = Multidimensional Body-Self Relations Questionnaire; CESD-R = Center for Epidemiological Studies Depression scale – Revised; AUDIT = Alcohol Use Disorders Identification Test; STICSA = State Trait Inventory for Cognitive and Somatic Anxiety; BEAQ = Brief Experiential Avoidance Questionnaire. 1Odds Ratios: Behavior coded as 1 = present and 0 = absent 2Controlling for other RES scales and sex only. 2Controlling for other RES scales and sex only. *p < .001; p <.01; * p <.05. Figures Page 24/26 Figure 1 Scree plot of obtained Eigenvalues superimposed on the results of a parallel analysis using 1000 random datasets indicating that three factors should be extracted Figure 1 Scree plot of obtained Eigenvalues superimposed on the results of a parallel analysis using 1000 random datasets indicating that three factors should be extracted Page 25/26 Page 25/26 Results of confirmatory factor analysis indicating a three-factor solution for the Reactive Exercise Scale Note. Numbers in boxes indicate item numbers and correspond to those in Table 1. Error covariance was freely estimated for similarly worded items. Page 26/26
https://openalex.org/W2052554989	http://repository.ajou.ac.kr/bitstream/201003/2587/3/20604967.pdf	English	null	Impact of Body Mass Index on the relationship of epicardial adipose tissue to metabolic syndrome and coronary artery disease in an Asian population	Cardiovascular diabetology	2,010	cc-by	5,999	Abstract Background: In a previous study, we demonstrated that the thickness of epicardial adipose tissue (EAT), measured by echocardiography, was increased in patients with metabolic syndrome (MS) and coronary artery disease (CAD). Several studies on obese patients, however, failed to demonstrate any relationship between EAT and CAD. We hypothesized that body mass index (BMI) affected the link between EAT and MS and CAD. Methods: We consecutively enrolled 643 patients (302 males, 341 females; 59 ± 11 years), who underwent echocardiography and coronary angiography. The EAT thickness was measured on the free wall of the right ventricle at the end of diastole. All patients were divided into two groups: high BMI group, ≥27 kg/m2 (n = 165), and non-high BMI group, < 27 kg/m2 (n = 478). Results: The median and mean EAT thickness of 643 patients were 3.0 mm and 3.1 ± 2.4 mm, respectively. In the non- high BMI group, the median EAT thickness was significantly increased in patients with MS compared to those without MS (3.5 vs. 1.9 mm, p < 0.001). In the high BMI group, however, there was no significant difference in the median EAT thickness between patients with and without MS (3.0 vs. 2.5 mm, p = 0.813). A receiver operating characteristic (ROC) curve analysis predicting MS revealed that the area under the curve (AUC) of the non-high BMI group was significantly larger than that of the high BMI group (0.659 vs. 0.506, p = 0.007). When compared to patients without CAD, patients with CAD in both the non-high and high BMI groups had a significantly higher median EAT thickness (3.5 vs. 1.5 mm, p < 0.001 and 4.0 vs. 2.5 mm, p = 0.001, respectively). However, an ROC curve analysis predicting CAD revealed that the AUC of the non-high BMI group tended to be larger than that of the high BMI group (0.735 vs. 0.657, p = 0.055). Conclusions: While EAT thickness was significantly increased in patients with MS and CAD, the power of EAT thickness to predict MS and CAD was stronger in patients with BMI < 27 kg/m2. These findings showed that the measurement of EAT thickness by echocardiography might be especially useful in an Asian population with a non-high BMI, less than 27 kg/m2. reliable imaging indicator of VAT. * Correspondence: shinjh@ajou.ac.kr 1 Department of Cardiology, Ajou University School of Medicine, Suwon, Korea † Contributed equally Full list of author information is available at the end of the article © 2010 Park et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons At- tribution 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. ORIGINAL INVESTIGATION ORIGINAL INVESTIGATION Abstract It is widely known that EAT thickness measured by echocardiography has a good correlation with abdominal adipose tissue measured by computed tomography (CT) and magnetic resonance imaging (MRI) [2,3]. CARDIO VASCULAR DIABETOLOGY CARDIO VASCULAR DIABETOLOGY Park et al. Cardiovascular Diabetology 2010, 9:29 http://www.cardiab.com/content/9/1/29 CARDIO VASCULAR DIABETOLOGY Original investigation Impact of Body Mass Index on the relationship of epicardial adipose tissue to metabolic syndrome and coronary artery disease in an Asian population Jin-Sun Park†1, Sung-Gyun Ahn†2, Jung-Won Hwang†1, Hong-Seok Lim†1, Byoung-Joo Choi†1, So-Yeon Choi†1, Myeong-Ho Yoon†1, Gyo-Seung Hwang†1, Seung-Jea Tahk†1 and Joon-Han Shin*1 Background Central obesity (visceral adipose tissue) correlates strongly with the development of metabolic syndrome (MS) and coronary artery disease (CAD) [1]. There are ways to estimate visceral adipose tissue (VAT). Recently, there are several reports that proposed echocardio- graphic epicardial adipose tissue (EAT) as an easy and In our previous study, the EAT thickness increased lin- early as the MS score increased and the EAT was thicker in patients with MS than in those without MS. The EAT was thicker in patients with significant CAD than in those without CAD. We suggested that EAT thickness measured by echocardiography was a good surrogate Park et al. Cardiovascular Diabetology 2010, 9:29 http://www.cardiab.com/content/9/1/29 Park et al. Cardiovascular Diabetology 2010, 9:29 http://www.cardiab.com/content/9/1/29 Page 2 of 8 marker for MS and CAD [3,4]. In another study, EAT thickness was also related to MS and the presence and extent of CAD [5]. as the number of criteria present. Adjusting values specif- ically for an Asian population, central obesity was defined as waist circumference of ≥90 cm in men and ≥80 cm in women [16]. Upon quantitative analysis of the coronary angiograms, significant CAD was considered to be the presence of one or more stenoses, ≥50% in diameter, of a major epicardial vessel. The major epicardial vessels were defined as the left main coronary artery that divides into the left anterior descending and circumflex branches, and the right coronary artery. [ ] Several groups have failed to demonstrate the correla- tion between EAT and CAD. Chaowalit et al. reported that EAT thickness is not associated with the presence, extent or severity of CAD in patients [6]. Gorter et al. reported that neither pericardial fat nor visceral abdomi- nal fat correlated with CAD in patients with body mass index (BMI) ≥25 kg/m2 [7]. In these reports, the subjects were relatively obese compared with the subjects in our previous study. In our study, the mean BMI of the sub- jects was 25.2 ± 3.1 kg/m2. In the report of Chaowalit et al., the mean BMI of the subjects was 28.8 ± 5.5 kg/m2 [6]. The inconsistent results regarding the correlation between EAT and CAD might be due to differences in BMI of the study population. In one study, the correlation between pericardial fat and coronary plaque was seen in the non-obese patients but not in the overweight patients [8]. Background To evaluate the correlation between pericardial fat and CAD, it has been suggested that it is necessary to divide study subjects according to the degree of obesity, as obesity itself is frequently associated with various met- abolic and circulatory factors and is known to be an inde- pendent risk factor for CAD [9]. Mazur et at. failed to demonstrate the correlation between EAT and MS. In that report, the subjects included obese children with BMI > 97 percentile as defined by the International Obe- sity Task Force [10,11]. In 2004, Kip et al. reported that the clinical effects of VAT on patients with MS differed between the normal BMI group and the obese group. In that study, the patients with MS and normal BMI had poorer clinical outcome compared to the patients with MS who were obese [12]. We hypothesized that the pre- dicting ability of EAT thickness for MS and CAD is affected by BMI. Measurement of echocardiographic epicardial adipose tissue Two-dimensional transthoracic echocardiography was performed. Recordings of six cycles of the two-dimen- sional parasternal long-axis were obtained. We enlarged each view for better visualization and accurate measure- ment of EAT thickness. The measurement of EAT thick- ness was done offline through the DICOM system and performed on the free wall of the right ventricle (RV) in the still image of a 2-D echocardiogram at end diastole on the parasternal long-axis. We preferred the area of above the RV to measure EAT thickness, because this area is recognized as having the thickest EAT layer. In addition, the parasternal long-and short-axis views allow the most accurate measurement of EAT thickness with optimal cursor beam orientation in each view. We measured the thickest point of EAT in each cycle. The average value of the EAT thickness was calculated (Figure 1). Coronary angiography Quantitative coronary angiographic analysis was per- formed by one experienced technician who was unaware of the patients' clinical information. Angiographic data were analyzed for the presence of CAD. Figure 1 Echocardiographic measurement of epicardial adipose tissue thickness. Parasternal long-axis view at the mitral valve level. EAT, epicardial adipose tissue; RV, right ventricle Statistical analysis variability of the EAT measurement, indicating good reproducibility. The SPSS 12.0 (SPSS inc., Chicago, Illinois, USA) statisti- cal software package was used for all calculations. Data are shown as the mean ± standard deviation (SD) and the median values for continuous variables and as percent- ages for categorical variables. p < 0.05 indicates statistical significance. We compared the characteristics of patients who had BMI ≥27 kg/m2 (high BMI group) to those with BMI < 27 kg/m2 (non-high BMI group). Comparisons of continuous variables were performed using the unpaired Student t test. As the skewness of the EAT thickness was 0.749 (p < 0.001) using the Kolmogrov-Smirnov test, comparisons of the EAT thickness according to the pres- ence of MS in the high and non-high BMI groups and the presence of CAD in 2 groups were performed using the Wilcoxon rank-sum test rather than the parametric test. Comparisons of the EAT thickness according to the MS score were done using the Kruskal-Wallis test. A receiver operation characteristic (ROC) curve analysis was used to assess the discrimination of MS and CAD based on the EAT thickness according to the BMI. Medcalc 10.4.8 (Medcalc software inc., Mariakerke, Belgium) statistical software was used for comparing the area under curve (AUC) of the ROC curve between groups. Clinical characteristics Clinical characteristics according to BMI are summarized in Table 1. Seventy-four percent of patients were included in the non-high BMI group. The non-high BMI group consisted of 478 patients (252 males) with a mean age of 59 ± 11 years. Forty-nine percent of non-high BMI group met the criteria of the MS. The high BMI group was con- sisted of 165 patients (80 males) with a mean age of 59 ± 11 years. Eighty-one percent of the high BMI group was diagnosed with MS. Epicardial adipose tissue thickness as a predictor of coronary artery disease according to body mass index The median EAT thickness was significantly increased in patients with CAD compared to patients without CAD (1.7 vs. 3.5 mm, p < 0.001). When compared to subjects without CAD, patients with CAD in both the non-high and high BMI groups had a median EAT thickness that was significantly increased (3.5 vs. 1.5 mm, p < 0.001 and 4.0 vs. 2.5 mm, p = 0.001, respectively, Figure 5). In addi- tion to well-known CAD risk factors, such as age, smok- ing, hypertension, and diabetes, EAT thickness was an independent factor associated with CAD in both groups (Table 2). The ROC curves of the EAT thickness in pre- dicting CAD yielded an AUC of 0.657 in the non-high BMI group and 0.735 in the high BMI group. When com- paring the AUC of the 2 groups, the ROC curve analysis predicting CAD revealed that the AUC of the BMI < 27 kg/m2 group tended to be larger than that of the BMI ≥27 kg/m2 (p = 0.055, Figure 6). Compared to the non-high BMI group, subjects in the high BMI group had significantly higher rates of hyper- tension, diabetes and metabolic syndrome and higher lev- els of total cholesterol, LDL-cholesterol and triglycerides. The median and mean EAT thickness of 643 patients were 3.0 mm and 3.1 ± 2.4 mm, respectively. In the non- high BMI group, the median and mean EAT thickness were 2.8 mm and 3.0 ± 2.4 mm, respectively. In the high BMI group, the median and mean EAT thickness were 3.0 mm and 3.5 ± 2.4 mm, respectively. The median EAT thickness was significantly increased in the high BMI group compared to that of the non-high BMI group (3.0 vs. 2.8 mm, p = 0.01). The absolute values of the mean paired differences were 0.0200 ± 0.39637, p = 0.773 and 0.0033 ± 0.46866, p = 0.969 for inter- and intraobserver Study population The EAT thickness was measured consecutively in 643 patients (mean age 59 ± 11 years; 302 males, 341 females) who underwent their first coronary angiography due to chest pain. The medical records of all patients were retro- spectively reviewed after informed consent was obtained from patients. EAT has been proposed as a source of sev- eral inflammatory mediators [13-15]. We tried to exclude the acute systemic inflammatory effect to avoid con- founding the role of EAT and diagnosis of MS. We excluded patients from the study if they had any of the following: active inflammation, a history of prior revascu- larization, heart failure, cardiomyopathy or acute myo- cardial infarction. We identified MS if there were more than 3 of 5 criteria based on the updated Adult Treatment Panel III (ATP III) guidelines. The MS score was defined Figure 1 Echocardiographic measurement of epicardial adipose tissue thickness. Parasternal long-axis view at the mitral valve level. EAT, epicardial adipose tissue; RV, right ventricle Park et al. Cardiovascular Diabetology 2010, 9:29 http://www.cardiab.com/content/9/1/29 Park et al. Cardiovascular Diabetology 2010, 9:29 http://www.cardiab.com/content/9/1/29 Page 3 of 8 Epicardial adipose tissue thickness as a predictor of metabolic syndrome according to body mass index The median EAT thickness was significantly increased in patients with MS compared to those without MS (3.3 vs. 2.0 mm, p < 0.001). EAT thickness also increased with an increase in the number of MS components (p < 0.001). In the non-high BMI group, the median EAT thickness was significantly increased in patients with MS compared to those without MS (3.5 vs. 1.9 mm, p < 0.001, Figure 2). The median EAT thickness values for those with 0, 1, 2, 3, 4, or 5 MS components were 1.3, 1.7, 2.0, 3.5, 3.5, and 3.2 mm, respectively. The median EAT thickness signifi- cantly increased with an increase in the number of MS components (p < 0.001, Figure 3-A). In the high BMI group, the median EAT thickness was not significantly increased in patients with MS compared to those without MS (3.0 vs. 2.5 mm, p = 0.813, Figure 2). The median EAT thickness values for subjects with 0, 1, 2, 3, 4, or 5 MS components were 2.5, 2.3, 3.3, 3.0, 3.0, and 4.5 mm, respectively. The median EAT thickness did not increase with an increase in the number of MS compo- nents (p = 0.121, Figure 3-B). One sample paired t test was performed to evaluate inter- and intraobserver variability in the measurement of EAT from 30 randomly selected subjects. The ROC curves of the EAT thickness in predicting MS yielded an AUC of 0.659 in the non-high BMI group and 0.506 in the high BMI group. When comparing the AUC of the 2 groups, the AUC to predict MS was significantly higher in the non-high BMI group compared to the high BMI group (p = 0.007, Figure 4). Discussion The present study demonstrated that the power of EAT thickness measured by echocardiography to predict MS Park et al. Cardiovascular Diabetology 2010, 9:29 http://www.cardiab.com/content/9/1/29 Page 4 of 8 Table 1: Baseline characteristics according to body mass index (n = 643) Characteristics BMI < 27 kg/m2 (n = 478) BMI ≥ 27 kg/m2 (n = 165) p Male, No 252 (53%) 80 (48%) NS Age (year-old) 59 ± 11 59 ± 11 NS EAT thickness (mm) Mean ± SD 3.0 ± 2.4 3.5 ± 2.4 0.01 Median 2.8 3.0 0.01 Body Mass Index (kg/m2) 23.7 ± 2.4 29.0 ± 2.1 < 0.001 Waist circumference (cm) Male 87.0 ± 8.0 97.1 ± 7.5 < 0.001 Female 84.7 ± 9.1 95.0 ± 7.6 < 0.001 Total cholesterol (mg/dl) 170.6 ± 35.3 182.32 ± 39.2 0.001 LDL cholesterol (mg/dl) 97.0 ± 30.0 104.4 ± 33.7 0.025 Triglyceride (mg/dl) 142.1 ± 83.3 183.1 ± 109.5 < 0.001 HDL cholesterol (mg/dl) Male 44.0 ± 10.7 40.3 ± 9.2 0.013 Female 48.3 ± 12.7 47.1 ± 11.3 NS Fasting glucose (mg/dl) 115.0 ± 33.9 120.0 ± 43.4 NS Hypertension, No 313 (66%) 125 (76%) 0.012 Diabetes, No 295 (62%) 128 (78%) < 0.001 Smoking, No 156 (33%) 41 (25%) 0.048 Metabolic Syndrome, No 243 (49%) 133 (81%) < 0.001 Metabolic Score 2.5 ± 1.3 3.3 ± 1.0 < 0.001 Coronary Artery Disease, No 247 (52%) 74 (45%) NS Normal & minimal CAD 230 (48%) 91 (55%) NS 1 vessel 111 (23%) 31 (19%) NS 2 vessel 69 (14%) 24 (15%) NS 3 vessel 65 (14%) 19 (12%) NS Medication, No Aspirin 115 (24%) 37(22%) NS Beta blockers 49 (10%) 19 (12%) NS Calcium channel blockers 89 (19%) 33 (20%) NS ACE inhibitors 23 (5%) 4 (2%) NS AT II receptor blockers 58 (12%) 22 (13%) NS Statins 46 (10%) 22 (13%) NS Nitrates 51 (11%) 16 (10%) NS Diuretics 46 (10%) 18 (11%) NS EAT, epicardial adipose tissue; LDL, low-density lipoprotein; HDL, high-density lipoprotein; CAD, coronary artery disease; ACE, angiotensin converting enzyme; AT, angiotensin; SD, standard deviation; NS, not significant was stronger in patients with BMI < 27 kg/m2. Our results also demonstrated a strong correlation between EAT between EAT and CAD [3,6,7,17]. Discussion Recently Gorter et a reported that in patients with BMI < 27 kg/m2 increase Table 1: Baseline characteristics according to body mass index (n = 643) EAT, epicardial adipose tissue; LDL, low-density lipoprotein; HDL, high-density lipoprotein; CAD, coronary artery disease; ACE, angiotensin converting enzyme; AT, angiotensin; SD, standard deviation; NS, not significant between EAT and CAD [3,6,7,17]. Recently Gorter et al. reported that, in patients with BMI < 27 kg/m2, increased EAT volume and pericoronary fat volume as measured by CT were associated with CAD and extensive coronary artery calcium (CAC) [7]. Although we used a different was stronger in patients with BMI < 27 kg/m2. Our results also demonstrated a strong correlation between EAT thickness and CAD, especially in patients with a non-high BMI. Our study might explain previous inconsistent results from various studies regarding the correlation Park et al. Cardiovascular Diabetology 2010, 9:29 http://www.cardiab.com/content/9/1/29 Page 5 of 8 Figure 2 Distribution of the thickness of epicardial adipose tissue according to body mass index and metabolic syndrome. Figure 4 ROC curve analysis to demonstrate the discriminatory power of the thickness of epicardial adipose tissue in the diagno- sis of metabolic syndrome according to body mass index. AUC, the area under the curve Figure 2 Distribution of the thickness of epicardial adipose tissue according to body mass index and metabolic syndrome. Figure 4 ROC curve analysis to demonstrate the discriminatory power of the thickness of epicardial adipose tissue in the diagno- sis of metabolic syndrome according to body mass index. AUC, the area under the curve way to evaluate EAT, using echocardiography to measure EAT thickness, the results were consistent with those of Gorter et al. In addition to these findings, we demon- strated that the predicting value of EAT thickness for MS might be more useful in patients with a non-high BMI. The results of our study suggest that different standards of EAT thickness according to BMI are needed to predict MS and CAD. way to evaluate EAT, using echocardiography to measure EAT thickness, the results were consistent with those of Gorter et al. In addition to these findings, we demon- strated that the predicting value of EAT thickness for MS might be more useful in patients with a non-high BMI. The results of our study suggest that different standards of EAT thickness according to BMI are needed to predict MS and CAD. Discussion In the present study, we used 27 kg/m2 as the cut-off point; any number equal to 27 and above was the BMI for obesity. The relative percentage of body fat at different BMIs clearly varies within populations. The correspond- ing cut-off point for obesity, based on the assumption that the percentage of body fat in the Asian population is the same as the percentage of body fat in the Western population, was lower than the existing WHO cut-off point for obesity, 30 kg/m2 [18]. Western studies have reported that a BMI ≥ 30 kg/m2 group has a greater risk of death caused by cardiovascular diseases [19,20]. Some recent studies on Asian populations showed that low BMI influences cardiovascular diseases [18,21]. In 2007, the Kangwha Cohort Study reported that the risk of cardio- vascular diseases was significantly increased in the BMI ≥ 27 kg/m2 group in the Korean population [22]. This study included the Korean population, so we decided that the cut-off point for obesity was 27 kg/m2 instead of 30 kg/ m2. p g y p p the same as the percentage of body fat in the Western population, was lower than the existing WHO cut-off point for obesity, 30 kg/m2 [18]. Western studies have reported that a BMI ≥ 30 kg/m2 group has a greater risk of death caused by cardiovascular diseases [19,20]. Some recent studies on Asian populations showed that low BMI influences cardiovascular diseases [18,21]. In 2007, the Kangwha Cohort Study reported that the risk of cardio- vascular diseases was significantly increased in the BMI ≥ 27 kg/m2 group in the Korean population [22]. This study included the Korean population, so we decided that the cut-off point for obesity was 27 kg/m2 instead of 30 kg/ m2. Figure 3 Distribution of the thickness of epicardial adipose tissue according to body mass index and metabolic score. (A) Non-high BMI group. (B) High BMI group. A B Figure 5 Distribution of the thickness of epicardial adipose tissue according to body mass index and coronary artery disease. A B Figure 5 Distribution of the thickness of epicardial adipose tissue according to body mass index and coronary artery disease. Figure 5 Distribution of the thickness of epicardial adipose tissue according to body mass index and coronary artery disease. Figure 3 Distribution of the thickness of epicardial adipose tissue according to body mass index and metabolic score. (A) Non-high BMI group. Discussion (B) High BMI group. Figure 3 Distribution of the thickness of epicardial adipose tissue according to body mass index and metabolic score. (A) Non-high BMI group. (B) High BMI group. Figure 5 Distribution of the thickness of epicardial adipose tissue according to body mass index and coronary artery disease. Park et al. Cardiovascular Diabetology 2010, 9:29 http://www.cardiab.com/content/9/1/29 Page 6 of 8 Figure 6 ROC curve analysis to demonstrate the discriminatory power of the thickness of epicardial adipose tissue in the diagno- sis of coronary artery disease according to body mass index. AUC, the area under the curve and metabolism of pro- and anti-inflammatory mediators secreted by EAT have been linked to insulin sensitivity [23]. EAT has been shown to express a pathogenic mRNA profile of pro-inflammatory adipokines in patients with CAD [13]. One study showed that expression of adi- ponectin, an anti-inflammatory adipokine, in EAT was significantly lower in patients with CAD compared to those without CAD [14]. EAT thickness has also been shown to be related to markers of insulin resistance and inflammation [2]. For example, one study demonstrated that obesity leads to adipocyte hypertrophy, which increases the secretion of pro-inflammatory adipokines and decreases the secretion of anti-inflammatory adipok- ines by EAT [23]. The change of EAT thickness by obesity might have obscured the difference between patients with and without CAD, or with and without MS, in the high BMI group of our study. Figure 6 ROC curve analysis to demonstrate the discriminatory power of the thickness of epicardial adipose tissue in the diagno- sis of coronary artery disease according to body mass index. AUC, the area under the curve The second possible explanation for the weak correla- tion between the EAT thickness and presence of MS and CAD in the high BMI group, might be the different pro- portion of the EAT to total amount of VAT according to BMI. Patients with high BMI generally have more VAT, which includes EAT. In the high BMI group, the EAT might make up a smaller proportion of the total VAT Several biomolecular studies in humans have shown that EAT is metabolically active and an important source of both pro-inflammatory adipokines, such as tumor necrosis factor-α, interleukin 1, interleukin 6 and nerve growth factor, and anti-inflammatory adipokines, such as adiponectin and adrenomedullin [13-15]. References 1. National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III): Third Report of the National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III) final report. Circulation 2002, 106:3143-3421. 1. National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III): Third Report of the National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III) final report. Circulation 2002, 106:3143-3421. 2. Iacobellis G, Ribaudo MC, Assael F, Vecci E, Tiberti C, Zappaterreno A, Di Mario U, Leonetti F: Echocardiographic epicardial adipose tissue is related to anthropometric and clinical parameters of metabolic syndrome: a new indicator of cardiovascular risk. J Clin Endocrinol Metab 2003, 88:5163-5168. 2. Iacobellis G, Ribaudo MC, Assael F, Vecci E, Tiberti C, Zappaterreno A, Di Mario U, Leonetti F: Echocardiographic epicardial adipose tissue is related to anthropometric and clinical parameters of metabolic syndrome: a new indicator of cardiovascular risk. J Clin Endocrinol Metab 2003, 88:5163-5168. 3. Ahn SG, Lim HS, Joe DY, Kang SJ, Choi BJ, Choi SY, Yoon MH, Hwang GS, Tahk SJ, Shin JH: Relationship of epicardial adipose tissue by echocardiography to coronary artery disease. Heart 2008, 94:e7. 3. Ahn SG, Lim HS, Joe DY, Kang SJ, Choi BJ, Choi SY, Yoon MH, Hwang GS, Tahk SJ, Shin JH: Relationship of epicardial adipose tissue by echocardiography to coronary artery disease. Heart 2008, 94:e7. 4. Baik SH, Ahn SG, Choi JH, Koh BR, Yoo JH, Kang SJ, Choi BJ, Choi SY, Yoon MH, Tahk SJ, Shin JH: The relationship of epicardial adipose tissue to metabolic syndrome and cardiovascular risk factors. Korean J Med 2007, 72:290-297. 4. Baik SH, Ahn SG, Choi JH, Koh BR, Yoo JH, Kang SJ, Choi BJ, Choi SY, Yoon MH, Tahk SJ, Shin JH: The relationship of epicardial adipose tissue to metabolic syndrome and cardiovascular risk factors. Korean J Med 2007, 72:290-297. 5. Eroglu S, Sade LE, Yildirir A, Bal U, Ozgul AS, Bozbas H, Anydinalp A, Muderrisoglu H: Epicardial adipose tissue thickness by echocardiography is a marker for the presence and severity of coronary artery disease. Nutr Metab Cardiovasc Dis 2009, 19(3):211-217. 6. Author Details 1Department of Cardiology, Ajou University School of Medicine, Suwon, Korea and 2Division of Cardiology, Yonsei University Wonju College of Medicine, Wonju, Korea There are several limitations to the present study. First, the study population was highly selective for those who underwent their first coronary angiography due to chest pain. Therefore, the results of the study could not be applied to the general population. Also, it was a cross- sectional study, not a cohort study. In a community-based prospective cohort study, it was reported that pericardial fat predicts incident coronary heart disease independent of conventional risk factors. They evaluated the correla- tion between pericardial fat and CAD without dividing study subjects according to the degree of obesity [24]. A prospective cohort study might be needed to elucidate the different predicting power of EAT for MS and CAD according to BMI in the general population. Second, EAT thickness by echocardiography does not exactly represent the amount of total EAT. Even though echocardiography is not the optimal method for quantification of EAT, our previous study showed that EAT thickness measured by echocardiography has a good correlation with the total amount of EAT [25]. EAT is true visceral fat deposited around the heart. Epicardial, mesenteric, and omental fat all share the same origin from the splanchnopleurituc mesoderm associated with the gut [26]. EAT thickness measured by echocardiography provides a sensitive and specific measurement of true visceral fat, avoiding the confounding effect caused by subcutaneous fat [27]. As echocardiography is frequently performed in high-risk cardiac patients, EAT thickness measured by echocar- diography may be readily available at no extra cost. Therefore, EAT thickness by echocardiography could be applied as an easy and reliable imaging indicator of VAT without radiation exposure [2,3]. Authors' contributions All authors participated in the design and coordination of the study, reviewed the analysis and took part in writing the manuscript. They also read and approved the final manuscript. References Chaowalit N, Somers VK, Pellikka PA, Rihal CS, Lopez-Jimenez F: Subepicardial adipose tissue and the presence and severity of coronary artery disease. Atherosclerosis 2006, 186(2):354-359. 7. Gorter PM, de Vos AM, van der Graaf Y, Stella PR, Doevendans PA, Meijs MF, Prokop M, Visseren FL: Relation of epicardial and pericoronary fat to coronary atherosclerosis and coronary artery calcium in patients undergoing coronary angiography. Am J Cardiol 2008, 102:380-385. 8. Yong HS, Kim EJ, Seo HS, Kang EY, Kim YK, Woo OH, Han H: Pericardial fat is more abundant in patients with coronary atherosclerosis and even in the non-obese patients: evaluation with cardiac CT angiography. Int J Cardiovasc Imaging 2010, 26:53-62. 9. Taguchi R, Takasu J, Itani Y, Yamamoto R, Yokoyama K, Watanabe S, Masuda Y: Pericardial fat accumulation in men as a risk factor for coronary artery disease. Atherosclerosis 2001, 157:203-209. 10. Mazur A, Ostański M, Telega G, Malecka-Tendera E: Is epicardial fat tissue a marker of metabolic syndrome in obese children? Atheroscleorsis 2010 in press. Competing interests h h d l h The authors declare that they have no competing interests. Acknowledgements We express our gratitude to Minje Sung, PhD in Ajou University, School of Busi- ness Administration for help with statistical analysis and SPSS programming. Discussion The regulation Table 2: Multiple logistic analysis of coronary artery disease risk factors (A) Non-high BMI group (n = 478) Risk factors Odds ratio (95% CI) p Age 2.847 (1.603-5.057) < 0.001 Male 45 ≥ years Female 55 ≥ years Smoking 1.576 (1.014-2.448) 0.043 Hypertension 2.115 (1.385-3.230) < 0.001 Diabetes mellitus 2.139 (1.282-3.569) 0.004 EAT thickness (≥3 mm) 3.504 (2.297-5.346) < 0.001 (B) High BMI group (n = 165) Risk factors Odds ratio (95% CI) p Age 6.531 (2.089-20.420) < 0.001 Male 45 ≥ years Female 55 ≥ years Smoking 1.775 (0.763-4.129) 0.183 Hypertension 1.276 (0.6-2.712) 0.527 Diabetes mellitus 2.687 (1.222-5.911) 0.014 EAT thickness (≥3 mm) 2.341 (1.136-4.827) 0.021 EAT, epicardial adipose tissue; CI, confidence interval Table 2: Multiple logistic analysis of coronary artery disease risk factors Page 7 of 8 Page 7 of 8 Page 7 of 8 Park et al. Cardiovascular Diabetology 2010, 9:29 http://www.cardiab.com/content/9/1/29 compared to the non-high BMI group. In the high BMI group, the EAT thickness by echocardiography might not be representative of the total VAT owing to its smaller proportion of the total VAT. That reason might attenuate the predictive value of EAT thickness by echocardiogra- phy for MS and CAD in the high BMI group. Further study is necessary to clarify the different roles of EAT according to BMI. Considering racial and gender differ- ences in addition to BMI, further study will be necessary to set the standards of EAT thickness by echocardiogra- phy. Park et al. Cardiovascular Diabetology 2010, 9:29 http://www.cardiab.com/content/9/1/29 14. Iacobellis G, Pistilli D, Gucciardo M, Leonetti F, Miraldi F, Brancaccio G, Gallo P, di Gioia CR: Adiponectin expression in human epicardial adipose tissue in vivo is lower in patients with coronary artery disease. Cytokine 2005, 29:251-255. 14. Iacobellis G, Pistilli D, Gucciardo M, Leonetti F, Miraldi F, Brancaccio G, Gallo P, di Gioia CR: Adiponectin expression in human epicardial adipose tissue in vivo is lower in patients with coronary artery disease. Cytokine 2005, 29:251-255. 15. Iacobellis G, Barbaro G: The double role of epicardial adipose tissue as pro- and anti-inflammatory organ. Horm Metab Res 2008, 40(7):442-445. 15. Iacobellis G, Barbaro G: The double role of epicardial adipose tissue as pro- and anti-inflammatory organ. Horm Metab Res 2008, 40(7):442-445. 16. Grundy SM, Cleeman JI, Daniels SR, Donato KA, Eckel RH, Franklin BA, Gordon DJ, Krauss RM, Savage PJ, Smith SC Jr, Spertus JA, Fernando Costa: Diagnosis and management of the metabolic syndrome: an American Heart Association/National Heart, Lung, and Blood Institute scientific statement. Circulation 2005, 112:2735-2752. 16. Grundy SM, Cleeman JI, Daniels SR, Donato KA, Eckel RH, Franklin BA, Gordon DJ, Krauss RM, Savage PJ, Smith SC Jr, Spertus JA, Fernando Costa: Diagnosis and management of the metabolic syndrome: an American Heart Association/National Heart, Lung, and Blood Institute scientific statement. Circulation 2005, 112:2735-2752. 17. Jeong JW, Jeong MH, Yun KH, Oh SK, Park EM, Kim YK, Rhee SJ, Lee EM, Lee J, Yoo NJ, Kim NH, Park JC: Echocardiographic epicardial fat thickness and coronary artery disease. Circ J 2007, 71:536-539. 17. Jeong JW, Jeong MH, Yun KH, Oh SK, Park EM, Kim YK, Rhee SJ, Lee EM, Lee J, Yoo NJ, Kim NH, Park JC: Echocardiographic epicardial fat thickness and coronary artery disease. Circ J 2007, 71:536-539. 18. WHO expert consultation: Appropriate body-mass index for Asian populations and its implications for policy and intervention strategies. Lancet 2004, 363(9403):157-163. 18. WHO expert consultation: Appropriate body-mass index for Asian populations and its implications for policy and intervention strategies. Lancet 2004, 363(9403):157-163. 19. Shaper AG, Wannamethee SG, Walker M: Body weight: implications for the prevention of coronary heart disease, stroke, and diabetes mellitus in a cohort study of middle aged men. BMJ 1997, 314:1311-1317. 20. Calle EE, Thun MJ, Petrelli JM, Rodriguez C, Heath CW Jr: Body-mass index and mortality in a prospective cohort of U.S. adults. NEJM 1999, 341:1097-1105. 21. Conclusions While EAT thickness was significantly increased in patients with MS and CAD regardless of BMI, the power of EAT to predict MS and CAD was stronger in patients with BMI < 27 kg/m2. These findings showed that the measurement of EAT thickness by echocardiography might be useful in patients with non-high BMI, especially one less than 27 kg/m2. 11. Cole TJ, Bellizzi MC, Flegal KM, Dietz WH: Establishing a standard definition for child overweight and obesity worldwide international survey. BMJ 2000, 320:1240-1243. 12. Kip KE, Marroquin OC, Kelley DE, Johnson BD, Kelsey SF, Shaw LJ, Rogers WJ, Reis SE: Clinical importance of obesity versus the metabolic syndrome in cardiovascular risk in women: a report from the Women's Ischemia Syndrome Evaluation (WISE) study. Circulation 2004, 17; 109(6):706-713. 13. Baker AR, Silva NF, Quinn DW, Harte AL, Pagano D, Bonser RS, Kumar S, McTernan PG: : Human epicardial adipose tissue expresses a pathogenic profile of adipocytokines in patients with cardiovascular disease. Cardiovasc Diabetol 2006, 5:1-7. Page 8 of 8 Park et al. Cardiovascular Diabetology 2010, 9:29 http://www.cardiab.com/content/9/1/29 Cui R, Iso H, Toyoshima H, Date C, Yamamoto A, Kikuchi S, Kondo T, Watanabe Y, Koizumi A, Wada Y, Inaba Y, Tamakoshi A, JACC Study Group: Body mass index and mortality from cardiovascular disease among Japanese men and women: the JACC study. Stroke 2005, 36(7):1377-1382. 22. Hong JS, Yi SW, Kang HC, Ohrr H: Body mass index and mortality in South Korean men resulting from cardiovascular disease: a Kangwha cohort study. Ann Epidemiol 2007, 17(8):622-627. 23. Karastergiou K, Mohamed-Ali V: The autocrine and paracrine roles of adipokines. Mol Cell Endocrinol 2010, 318:69-78. 23. Karastergiou K, Mohamed-Ali V: The autocrine and paracrine roles of adipokines. Mol Cell Endocrinol 2010, 318:69-78. 24. Ding J, Hsu FC, Harris TB, Liu Y, Kritchevsky SB, Szklo M, Ouyang P, Espeland MA, Lohman KK, Criqui MH, Allison M, Bluemke DA, Carr JJ: The association of pericardial fat with incident coronary heart disease: the Multi-Ethnic Study of Atherosclerosis (MESA). Am J Clin Nutr 2009, 90(3):499-504. 25. Hwang JW, Choi UJ, Ahn SG, Lim HS, Kang SJ, Choi BJ, Choi SY, Yoon MH, Hwang GS, Tahk SJ, Shin JH, Kang DK: Echocardiographic plains reflecting total amount of epicardial adipose tissue as risk factor of coronary artery disease. J Cardiovasc Ultrasound 2008, 16(1):17-22. 26. Ho E, Shimada Y: Formation of the epicardium studied with the scanning electron microscope. Dev Biol 1978, 66(2):579-585. 27. Iacobellis G, Singh N, Wharton S, Sharma AM: Substantial changes in epicardial fat thickness after weight loss in severely obese subjects. Obesity 2008, 16(7):1693-1697. doi: 10.1186/1475-2840-9-29 Cite this article as: Park et al., Impact of Body Mass Index on the relationship of epicardial adipose tissue to metabolic syndrome and coronary artery dis- ease in an Asian population Cardiovascular Diabetology 2010, 9:29 doi: 10.1186/1475-2840-9-29 Cite this article as: Park et al., Impact of Body Mass Index on the relationship of epicardial adipose tissue to metabolic syndrome and coronary artery dis- ease in an Asian population Cardiovascular Diabetology 2010, 9:29
https://openalex.org/W4229460366	https://aip.scitation.org/doi/pdf/10.1063/5.0089457	English	null	Establishing temperature from radiance of dynamically compressed metals	Journal of applied physics	2,022	cc-by	11,832	RESEARCH ARTICLE \| MAY 10 2022 Establishing temperature from radiance of dynamically compressed metals Articles You May Be Interested In Articles You May Be Interested In Shocked silica aerogel radiance transition J. Appl. Phys. (September 2022) The Development and Characterization of an Absolute Pyrometer Calibrated for Radiance Responsivity AIP Conference Proceedings (September 2003) Radiance American Journal of Physics (May 1963) Shocked silica aerogel radiance transition 24 October 2024 04:35:50 ABSTRACT Precise temperature determination is a significant challenge in extreme environments of dynamic compression studies. How can radiance measurements taken in high-pressure shock experiments constrain temperature in a meaningful and physically consistent way? Experiments maintaining sample compression against a transparent window can be tailored to present a uniform measurement area with uncertain spec- tral emissivity. We compare several methods to analyze radiance collected at multiple wavelengths, applying statistical methods and physical principles to improve temperature inference. With proper radiance collection and analysis, dynamic temperature uncertainties become com- parable to thermomechanical ambiguities of the emitting surface. © 2022 Author(s). All article content, except where otherwise noted, is licensed under a Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). https://doi.org/10.1063/5.0089457 024 04:35 AFFILIATIONS 1Los Alamos National Laboratory, Los Alamos, New Mexico 87545, USA 2Sandia National Laboratories, Albuquerque, New Mexico 87185, USA 1Los Alamos National Laboratory, Los Alamos, New Mexico 87545, USA 2Sandia National Laboratories, Albuquerque, New Mexico 87185, USA II. BACKGROUND This work describes experimental design guidelines for analyz- ing radiance measurements in dynamic compression experiments. Several pyrometric techniques are considered for temperature cal- culation when spectral emissivity is not measured. Statistical Establishing temperature from radiance of dynamically compressed metals Establishing temperature from radiance of dynamically compressed metals I. INTRODUCTION methods to constrain emissivity and bound temperature inference are presented, focusing on published radiance data.15 Analysis results are then compared with experiments where emissivity is directly measured. Understanding temperature is crucial to determine material equations of state (EOS), phase transitions, and chemical reactions under dynamic compression.1–3 Multi-wavelength optical pyrome- try has been applied in these experiments to infer dynamic temper- ature for more than half a century.4–9 Two primary techniques are employed to create an emissive radiant surface under dynamic loading. For initially transparent materials, the radiant surface is an opaque shock front emitting through the ambient material.4,6,10,11 Measuring shock states of optically opaque materials, such as metals, requires a different configuration. Generally, the studied material is affixed to a transparent optical window. The window material maintains non-zero stress, presenting a steady material composition in a uniform pressure–temperature state across the measurement interface area while itself remaining transparent under shock.7–9,12,13 Mechanical and thermal equilibration at the three-layer sample-bond-window interface must be carefully under- stood14 for proper interpretation of the results. Understanding temperature is crucial to determine material equations of state (EOS), phase transitions, and chemical reactions under dynamic compression.1–3 Multi-wavelength optical pyrome- try has been applied in these experiments to infer dynamic temper- ature for more than half a century.4–9 Two primary techniques are employed to create an emissive radiant surface under dynamic loading. For initially transparent materials, the radiant surface is an opaque shock front emitting through the ambient material.4,6,10,11 We focus on steady shock states in this discussion. Recent experiments have begun to investigate thermal phenomena of materials and surfaces subjected to shock–release, shock–release– reshock, and other complex pressure loading/unloading paths.16 Pyrometric temperature measurements under these conditions are challenging17 and are just beginning to be understood. In some experiments of this type, temperature can be inferred from the equilibration patterns expressed in time-dependent radiance traces.18 In others, the fundamental quantity of temperature cannot be well-determined and instead the observable quantity of time- dependent radiance is reported. Temperature analyses in these types of experiments will be presented elsewhere. Journal of Applied Physics ARTICLE scitation.org/journal/jap A. Radiometry and pyrometry Radiometry is the careful measurement of radiance (power per unit area per unit solid angle) J. Appl. Phys. 131, 185901 (2022); doi: 10.1063/5.0089457 © Author(s) 2022 J. Appl. Phys. 131, 185901 (2022); doi: 10.1063/5.0089457 131, 185901-1 Journal of Applied Physics scitation.org/journal/jap (1) cal- na- rial. unit e T (2) g. 1 ugh nif- two but on, ng. d as erse (3) ted ion (4) (5) (6) . ro- gth =1, mit, adi † Absolute uncertainties are more significant for small radiance d/ i i it th t l l FIG. 1. Spectral radiance (above) and uncertainty scaling (below) curves. The former [Eq. (2)] is evaluated at 750 and 1000–4000 K in 500 K steps. Scale factor curves [Eqs. (4) and (5)] are shown for 0.05 and 0.1–0.5. 24 October 2024 04:35:50 Lm ¼ ð wm(λ) dL dλ dλ (1) (1) in one or more spectral bands. Each radiance measurement is local- ized by response function wm(λ), which is defined by a combina- tion of optical coupling (fibers, etc.), filters, and detector material. All bands view a common spectral radiance dL=dλ (power per unit area per unit solid angle per unit wavelength). Pyrometry is radiometry of a thermal source at temperature T with emissivity ϵ, dL dλ ¼ ϵ(λ) c1 λ5 ec2=λT 1 ½ (2) (2) using radiation constants c1 and c2.19 The upper plot in Fig. 1 shows spectral radiance curves for several temperatures. Although dL=dλ increases with temperature at all wavelengths, there is signif- icant variation in that increase. Values increase by about two orders of magnitude on the long wavelength side of this plot but more than five decades on the short wavelength side. Forward calculation of band radiance is simple integration, but the reverse calculation of temperature is more challenging. Narrow band measurements, where w(λ) can be approximated as Dirac delta function, provide a conceptual framework for reverse calculations, 24 October 2024 04:35:50 T ¼ c2 λm 1 ln ( ϵmc1 Lmλ5 m þ 1) : (3) (3) 24 October 2024 04:35:50 24 October 2024 04:35:50 Given band emissivity 0 , ϵm 1, temperature can be calculated from measured band radiance Lm. © Author(s) 2022 A. Radiometry and pyrometry The sensitivity of that calculation is shown below: Lm T @T @Lm ¼ þ λmT c2 1 ec2=λmT h i , (4) ϵm T @T @ϵm ¼ λmT c2 1 ec2=λmT h i , (5) λm T @T @λm ¼ 5 λmT c2 1 ec2=λmT h i 1: (6) The lower plot in Fig. 1 shows the magnitude of Eqs. (4) and (5). Equations (3)–(6) lead to several immediate conclusions. The lower plot in Fig. 1 shows the magnitude of Eqs. (4) and (5). Equations (3)–(6) lead to several immediate conclusions. † While total radiance (integrated across all wavelengths) is pro- portional to T4, spectral radiance at any particular wavelength scales by much greater/lesser degrees. FIG. 1. Spectral radiance (above) and uncertainty scaling (below) curves. The former [Eq. (2)] is evaluated at 750 and 1000–4000 K in 500 K steps. Scale factor curves [Eqs. (4) and (5)] are shown for 0.05 and 0.1–0.5. † The lowest possible temperature calculation occurs when ϵm=1, also known as the radiance temperature. There is no upper limit, i.e., T ! 1 as ϵm ! 0. † Absolute uncertainties are more significant for small radiance and/or emissivity than at large values. † Fractional temperature uncertainty scales with fractional radi- ance/emissivity uncertainty as λmT=c2. With proper choice of measurement wavelength, that scaling can be reduced to 10%, constraining temperature more stringently than the calculation inputs. Although these conclusions are based on the narrow-band approxi- mation, similar results are observed for rigorous band integration. J. Appl. Phys. 131, 185901 (2022); doi: 10.1063/5.0089457 131, 185901-2 131, 185901-2 © Author(s) 2022 Journal of Applied Physics scitation.org/journal/jap FIG. 2. Top: Ambient Hagen–Rubens emissivity bounds for several representa- tive metals vs wavelength; bottom: emissivity bound for Fe, shown at ambient T, P (solid blue), T ¼ 3000 K, ambient P (dashed-dotted orange), ambient T, 150 GPa (dashed-dotted yellow), and 3000 K at 150 GPa (dashed blue). Note that at 3000 K and ambient pressure Fe is in the liquid phase, which may induce more complex changes in ϵ. 24 Precise temperature measurement is equally sensitive to rela- tive uncertainty in both radiance and emissivity. However, radiance uncertainty is manageable: careful experimental design can limit measured radiance to Planckian emission from a surface at a nearly uniform temperature. A. Radiometry and pyrometry Furthermore, spectral bands can be moved and/or widened to increase light collection, and electrical noise may be mitigated (within limits). We describe considerations for radiometric measurements, including measurement wavelengths, avoiding or identifying non-Planckian emission, and the measure- ment of emissivity in dynamic experiments in Appendix A. In most experiments, however, emissivity cannot be measured and is generally uncontrollable. An experiment with sound radi- ance collection design, but where emissivity is not measured, will see emissivity uncertainty dominate the uncertainty in calculated temperature. The standard method to address this issue is to perform simultaneous radiance measurements at different wave- lengths to gather more information. This is still an underdeter- mined system of M measurements with M þ 1 unknowns (M band emissivities plus temperature). Some further information or bound on emissivity is needed to obtain meaningful pyrometric tempera- ture from a radiance measurement. FIG. 2. Top: Ambient Hagen–Rubens emissivity bounds for several representa- tive metals vs wavelength; bottom: emissivity bound for Fe, shown at ambient T, P (solid blue), T ¼ 3000 K, ambient P (dashed-dotted orange), ambient T, 150 GPa (dashed-dotted yellow), and 3000 K at 150 GPa (dashed blue). Note that at 3000 K and ambient pressure Fe is in the liquid phase, which may induce more complex changes in ϵ. B. Metal emissivity Normal emissivity is related to normal-hemispherical reflec- tance as ϵ(λ; θ ¼ 0) ¼ 1 R(λ; 0, 2π), (7) (7) Figure 2 shows emissivity estimates for several metals at ambient conditions using Eq. (9). A highly conductive metal like Au has very low infrared emissivity, around 1%–5%. Less con- ductive metals—Sn, Ni, Cr, and Fe—have infrared emissivities of 10%–20%, while very poor conductors like Ti can be 30% emissive. Emissivity generally decreases with wavelength, specifically as λ1=2 in Eq. (9). Although that approximation is not valid for visible and near-infrared (NIR) wavelengths, similar trends are observed in ambient temperature,22 elevated temperature,23–27 and dynamic compression28–30 experiments. Metal emissivity generally varies continuously across the infrared and visible spectrum, with less than 0.001/nm variation at energies less than the interband transi- tions. Emissivity generally increases with decreasing wavelength, from wavelengths ≏5000 nm to well below 1000 nm, where electron collisions and interband transitions create more complex absorp- tion resonances. for an opaque material.20 That reflectance is governed by a materi- al’s intrinsic optical properties and surface geometry. For a specular material in vacuum, the Fresnel equation links emissivity to complex refractive index n þ ik, ϵ(λ; θ ¼ 0) ¼ 4n(λ) n2(λ) þ k2(λ) þ 2n(λ) þ 1 : (8) (8) Although it is clear that large values of n and/or k lead to low emis- sivity, broader trends are difficult to identify. Consider the Hagen–Rubens21 limit as a conceptual starting point for metal emissivity. ϵ 4 ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ πc0E0 σλ r : (9) (9) Broadly, when material phase remains constant, electrical con- ductivity decreases in predictable ways with temperature. The Bloch–Gruneisen model describes this in detail from quantum- mechanical considerations and reduces roughly to σ / 1 T for T much greater than 0 K. Measurements show that conductivity decreases at a rate slightly greater than linear, but for a given range of temperatures, a linear coefficient, α, can be used for an effective approximation Here, σ is electrical conductivity, c0 is the speed of light, and E0 is vacuum permittivity (to avoid confusion with emissivity). This approximation incorporates assumptions that electrical conductiv- ity is isotropic, and that electron relaxation time is much slower than the optical period. The former condition is satisfied for liquid and monovalent metals; for multivalent metals, agreement is worse. A. Radiance temperature and single wavelength calculations For instance, samples may be bonded to or deposited on an optical window, with radiometry performed through that window. The host index for common adhesives is easily 1.3–1.5, and optical windows are typically 1.3–1.8. Moreover, most metals quickly oxidize in air, so a thin dielectric layer is almost always formed during sample handling. Such oxides can have host index values of 1.8–2.5, drastically modifying emissivity from the vacuum prediction. For instance, samples may be bonded to or deposited on an optical window, with radiometry performed through that window. The host index for common adhesives is easily 1.3–1.5, and optical windows are typically 1.3–1.8. Moreover, most metals quickly oxidize in air, so a thin dielectric layer is almost always formed during sample handling. Such oxides can have host index values of 1.8–2.5, drastically modifying emissivity from the vacuum prediction. Generally, any measurement of radiance at a single wave- length, with a given spectral emissivity value, will calculate an apparent temperature. This value is accurate if all radiance is Planckian in origin and arises from an emitting surface with one temperature and emissivity. However, a measured L and assumed ϵ produce an apparent T for any quantity of collected light, quite separate from any notion of an emitting surface with a true temper- ature! If spectral emissivity is allowed to freely vary in analysis, then a single-wavelength radiance measurement can describe an infinite range of temperatures above the temperature calculated for ϵ ¼ 1. In the extreme conditions of dynamic shock compression, likelihood of measurement contamination by radiance of non- thermal origin limits accuracy of temperature determination, while uncertainty in dynamic ϵ inhibits precision of temperature determination. The less-than-perfect nature (roughness) of any real surface finish generally increases emissivity at most wavelengths. The precise nature of this effect is complex. An ambient spectral emis- sivity measurement of the particular target metal interface before an experiment will account for effects of surface roughness and further complications of spectral variation of emissivity due to vari- able surface chemistry and oxidation states. The simplest temperature calculation, applicable at any single wavelength, is the absolute lower bound on temperature: the assumption that ϵ ¼ 1. This value can often be used as a compari- son metric or sanity check for relative temperatures between similar experiments, or between multiple measurements of the same experiment, without additional assumptions. C. Emissivity modifications Equation (8) often fails to predict sample emissivity from the published refractive index data. Several factors contribute to this discrepancy, where calculations tend to be smaller than measured emissivities. Host medium, oxidation states, and surface roughness are briefly considered here. Samples emit more light into a host medium nh . 1 than in vacuum ϵ(λ) ¼ 4nh n(λ) n2(λ) þ k2(λ) þ 2nh n(λ) þ n2 h : (11) ϵ(λ) ¼ 4nh n(λ) n2(λ) þ k2(λ) þ 2nh n(λ) þ n2 h : (11) (11) B. Metal emissivity Additional collisions and interband transitions are ignored in this model, but such effects can only increase emissivity by providing absorption mechanisms. Equation (9), therefore, underestimates emissivity at short wavelengths and converges with actual emissiv- ity at long wavelengths. σ(T) ¼ σ0 1 1 þ α(T T0): (10) (10) 131, 185901-3 J. Appl. Phys. 131, 185901 (2022); doi: 10.1063/5.0089457 © Author(s) 2022 131, 185901-3 J. Appl. Phys. 131, 185901 (2022); doi: 10.1063/5.0089457 131, 185901-3 © Author(s) 2022 Journal of Applied Physics ARTICLE scitation.org/journal/jap † The windowing method, in which minimum and maximum expected emissivity values are estimated, and temperature bounds are calculated from them. † The windowing method, in which minimum and maximum expected emissivity values are estimated, and temperature bounds are calculated from them. Increase in pressure without increasing temperature or phase change generally increases electrical conductivity.31–34 The temperature-induced increase and pressure-induced decrease of emissivity will offset to some degree. For the case of Fe at ≏3000 K and ≏150 GPa pressure, the competing effects theoretically lead to moderate emissivity increase (Fig. 2 bottom pane). Note that at 3000 K at ambient pressure Fe is in its liquid phase, which may induce more complex changes in ϵ. † The Gaussian method, which assumes that the temperature probability is described by the calculation of temperature from a distribution for the emissivity, whose bounds are two-standard-deviation limits. This section considers the above techniques using the narrow band approximation. Each method is tested with measured radi- ance data from a recent study,15 where temperature was measured at the interface of a cerium sample attached to a lithium fluoride (LiF) window. A gun-driven flyer plate impacted on the far side of the cerium sample, imparting a supported shock wave in the Ce sample that breaks out into the LiF window. In experiment 2S-598, a steady interface pressure of 21:78 + 0:05 GPa was maintained for time t ¼ 22:8–23.0 μs after a brief (&10 ns) equilibration of the adhesive layer. Radiance temperatures are shown as a function of time, but other calculations are based on a steady-state average. The reported temperature for this experiment was 1993 + 84 K. A. Radiance temperature and single wavelength calculations To correctly identify this radiance temperature, it is also necessary to consider the limits of the radiometric measurement. The radiance tempera- ture calculated from the noise floor of the radiometry system estab- lishes an effective noise temperature for the measurement. Any measurement of radiance below this level will calculate the same noise temperature. Total radiometry system noise in a given experi- ment will produce an apparent temperature fluctuation about the mean signal value, even for a perfectly stable emitting surface. When the temperature changes slowly compared to the radiometry system bandwidth, this apparent fluctuation in temperature can be reduced with averaging over time, or it can be eliminated by taking the mean value over a stable period. Radiance values measured in our example experiment are given in Table I. From these © Author(s) 2022 C. The free graybody model measurements at five wavelengths, the noise temperatures vary from ≏800 to 1700 K depending on wavelength. The mean radi- ance temperatures are given in Table I. The least radiance tempera- ture is 1704 K, and so the radiance temperature method suggests T 1704 K. Radiance temperature values and noise temperature values (seen at far left, before shock breakout) are shown in Fig. 3. Two-color pyrometry analysis may be generalized to analyze measurements at more than two wavelengths, by fitting M radiance measurements at M wavelengths with radiance calculations using one T and one ϵ at all wavelengths. The fit varies ϵ between 0.01 and 1.00 and T between 0 and some reasonable ceiling; it then minimizes the deviation between calculated L(T, ϵ) and measured L at all measurement wavelengths. This approach is inherently unphysical for metal surfaces, where ambient emissivity varies con- tinuously with wavelength, generally in a way that roughly decreases with λ at NIR and visible wavelengths. The measurements of La Lone and collaborators9,39 show that dynamic emissivity further varies from ambient emissivity independently at each mea- surement wavelength. III. SIMPLE PYROMETRY ANALYSIS Assuming that radiometry is precise, the nature of the radiat- ing surface being measured is understood, and the measured radi- ance is thermal in the origin, spectral emissivity is the limiting factor in temperature measurement precision. In this section, we will examine five simple techniques that attempt to constrain dynamic emissivity and thus allow bounding of dynamic tempera- ture using measured radiance alone. † The radiance temperature, calculated from the assumption that emissivity is unity, and other bounding techniques based upon a single-wavelength measurement. g g † Two-color pyrometry, where the radiance is measured at two wavelengths, and the emissivity is assumed to be identical for both measurements. † The free graybody method, an extension of two-color pyrometry to M-colors. 131, 185901-4 J. Appl. Phys. 131, 185901 (2022); doi: 10.1063/5.0089457 131, 185901-4 © Author(s) 2022 Journal of Applied Physics ARTICLE scitation.org/journal/jap TABLE I. Steady state radiances in shot 2S-598. Radiance was measured in five bands in the near-infrared and visible spectrum. Minimum temperature for each band assume blackbody emission. measurements are made at two wavelengths, and emissivity is assumed to be identical at each wavelength. Two-color analysis fits a Planck curve [Eq. (3)] to the two measurements, allowing two variables, T and ϵ to freely vary. Temperatures calculated from shot 2S-598 using this method are shown in Table II. Channel number Spectral band (nm) Band radiance (W/m2 sr) Minimum temperatures (K)a (K)b 1 1445–2020 26, 910 ± 320 1704 1702 2 1194–1372 11, 160 ± 420 1814 1817 3 816–889 1682 ± 57 1798 1805 4 686–754 1061 ± 41 1871 1879 5 488–508 19.9 ± 5.0 1890 1900 aNarrow band approximation. bRigorous band integration. These results clearly demonstrate the difficulties associated with two-color pyrometry in dynamic studies of materials with unknown, spectrally varying emissivity. Any two finite radiance measurements lie along some Planck curve, regardless of whether the radiance is collected from a pure graybody emitter. Assuming measurement wavelengths are precisely known and radiance is pre- cisely measured, error arises from uncertainty in the graybody assumption. Error induced in a two-color pyrometry measurement by spectral variance in emissivity can be precisely calculated numerically or approximated analytically.37 B. Two-color pyrometry The simplest multi-wavelength pyrometric method to deter- mine temperature is two-color pyrometry.35–38 Radiance FIG. 3. Experimental radiance temperature (ϵ ¼ 1) values from shot 2S-598 in the literature Ce study15 vs time. Note the shock breakout at ≏22:7 μs and interface stress release beginning at ≏23:8 μs. Noise temperatures ranging from ≏800 to 1700 K are visible before breakout. Note that the Ch 1 detector is slower than the others, causing an apparent delay in radiance. As a test of the free graybody model, we calculate the best-fit T and ϵ for all five radiance measurements in shot 2S-598. The result is T ¼ 2026 K, ϵ ¼ 0:39, which agrees with the published data within uncertainty bounds. Figure 4 plots the radiance data and free graybody fit. By varying the radiance within uncertainty bounds of 15% and repeating the fit, we estimate the uncertainty on this calculation, T ¼ 2026 + 65 K. Despite the criticism above, this method appears to work reasonably well for the Ce surface in this experiment. The free graybody model can also be very useful in classes of experiments where two very different temperatures are present. This will be detailed in future work. E. The Gaussian method for multiple wavelengths The windowing method is arguably the simplest case of esti- mating spectral emissivity probability. It assumes equal probability of a maximum and minimum emissivity value and no further information. It should be noted that the mean reported tempera- ture is not the same as the temperature calculated from ϵ ¼ 1 2 (ϵH þ ϵL). Taking ϵH and ϵL as parameters of a spectral emissivity statistical distribution allows more sophisticated analysis. Perhaps, the simplest example is the use of emissivity bounds as the two-standard-deviation values in a Gaussian temperature distri- bution for each channel. The Gaussian method temperature, here denoted TGauss, is then calculated as the multiplication of the probability of each individual channel temperature distribution. Figure 5 demonstrates this calculation. The high and low uncer- tainty bounds are taken as the temperatures at which the tempera- ture probability drops to 5% on either side of the peak value. For a pure Gaussian distribution, this would be +2σ. With these asym- metric temperature distributions, it represents the values within which ≏90%–99% of the temperature probability distribution lies, varying somewhat for each calculation dependent upon agreement between measurement wavelengths. FIG. 4. Free graybody emissivity fit for shot 2S-598 in the literature Ce study. The Planck curve with T ¼ 2026 K and ϵ ¼ 0:39 at all wavelengths fits the data points with 4%–16% error. Horizontal bars depict the width of each band. Radiance uncertainties are smaller than the plotted circles. multiple-wavelength analysis that places somewhat physical limits on emissivity to resolve the underdetermined system. For each wavelength band, an upper (ϵH) and lower (ϵL) bound are posited for spectral emissivity. Each band’s measured radiance is individu- ally best-fit to the total radiance of a single Planck function with one value of T and one value of ϵ. The temperature at ϵ ¼ ϵH is the low bound T for that channel, and the temperature at ϵ ¼ ϵL is the high bound T for that channel. The reported individual tem- perature of each channel is the geometric mean of these two values. p g The individual temperature calculations of each measurement channel can be combined by different methods described in several studies.8,15,42,43 In the most recent of these works, Jensen et al. report temperature taken as the mean of the mean temperature of each channel. D. Emissivity windowing method Temperature precision can be improved by assigning limits to ϵ(λ). The emissivity windowing method.40,41 is a simple TABLE II. Calculated two-color temperatures for shot 2S-598. Temperature Required (K) emissivity Channels 1 and 2 2453 0.18 Channels 2 and 3 1857 0.76 Channels 3 and 4 2510 0.07 Channels 4 and 5 1936 0.76 TABLE II. Calculated two-color temperatures for shot 2S-598. FIG. 3. Experimental radiance temperature (ϵ ¼ 1) values from shot 2S-598 in the literature Ce study15 vs time. Note the shock breakout at ≏22:7 μs and interface stress release beginning at ≏23:8 μs. Noise temperatures ranging from ≏800 to 1700 K are visible before breakout. Note that the Ch 1 detector is slower than the others, causing an apparent delay in radiance. J. Appl. Phys. 131, 185901 (2022); doi: 10.1063/5.0089457 131, 185901-5 © Author(s) 2022 J. Appl. Phys. 131, 185901 (2022); doi: 10.1063/5.0089457 © Author(s) 2022 J. Appl. Phys. 131, 185901 (2022); doi: 10.1063/5.0089457 131, 185901-5 Journal of Applied Physics ARTICLE scitation.org/journal/jap FIG. 4. Free graybody emissivity fit for shot 2S-598 in the literature Ce study. The Planck curve with T ¼ 2026 K and ϵ ¼ 0:39 at all wavelengths fits the data points with 4%–16% error. Horizontal bars depict the width of each band. Radiance uncertainties are smaller than the plotted circles. decreased by narrowing the focused time period and smoothing the shortest wavelength data. When the emissivity bounds contain the true value and mea- sured radiances are free from contamination, the mean of TL and TH is similar at every wavelength. The window method in this case produces sensible, likely conservative error bars. However, it remains agnostic about the true nature of emissivity and so may produce an overly-conservative total temperature compared to a method that uses statistical distributions to combine multi- wavelength measurements. If emissivity is poorly bounded or radi- ance measurements are contaminated, the mean of TL and TH dis- agrees between channels, increasing the greatest TL and decreasing the least TH. In this case, the smallest TH and the largest TL cannot be used as bounds. The distance between these bounds actually narrows as disagreement increases, becoming over-constrained and requiring the use of the smallest TL and largest TH as described above, to avoid non-physical (too small) bounds. Generally, the resulting uncertainty bounds are ≏3%–6% for radiance measure- ments with good agreement and ≏8%–15% for radiance measure- ments with poor agreement. A. Spectral band integration Rigorous band integration Lm ¼ c1 ð wm(λ) ϵ(λ) λ5 ec2=λT 1 ½ dλ (12) (12) avoids the assumption that wm(λ) δ(λ λm). Optical filters have finite width, which is usually needed for sufficient power collection in high-speed measurements. Full integration avoids conceptual mistakes about when the narrow-band approximation is valid, e.g., is a 50 nm full-width half-maximum sufficiently narrow for visible and infrared pyrometry? Figure 6 shows response shapes for the five pyrometry channels in this work. Minimum temperature calculations are the first test of rigor- ous band integration. Results (Table I) are similar to, but not identical with, the narrow band approximation. Differences start at 2–3 K in the infrared and increase to 10 K in the visible. However, the spectral bandwidth varies drastically, starting at hundreds of nanometers in the longest wavelength channels and decreasing to tens of nanometers in the shortest wavelength channels. In this particular case, narrow-band analysis is accurate to about 0.5%. FIG. 5. Gaussian method temperature calculation for shot 2S-598. Top left: Gaussian P(ϵ) distribution used at all λ. Top center: T(ϵ) for Ch 1 L measure- ment. Top right: Probability of T, given L, P(ϵ). Bottom: Temperature probability for Ch 1–5 of the measurement, and multiplied total temperature probability, TGauss for all wavelengths. The mean of TGauss is 1967 K and the uncertainty bounds are 1923 and 2030 K. Figure 7 illustrates temperature bounds for the cerium mea- surement using rigorous band integration for emissivities 0.1–1. The weighted average temperature is 2115 K. This emissivity range affects the long wavelength measurements more strongly than the short wavelengths, consistent with the “go bluer” approach described in Appendix A. However, 0.1 emissivity is overly pessi- mistic for cerium, and that bias has an outsized effect on the infra- red channels. Symbols on the longest wavelength bound show probability calculation is an indicator of its quality. A narrow distri- bution with a greater peak magnitude indicates better agreement than a wide distribution with a lesser peak magnitude. Both ϵ 1 and ϵ 0 have some finite probability but are clearly unphysical. The former may have negligible effect but the latter may introduce an infinite temperature to the calculation. FIG. 6. Spectral bands in experiment 2S-598. A 1550 nm notch in the longest wavelength channel blocks non-thermal light from fiber interferometry measurements. A. Spectral band integration Using the Gaussian method, the average temperature for shot 2S-598 is 1967þ63 44 K. This agrees with the windowing method while giving somewhat improved uncertainty bounds. It is difficult to evaluate the physical meaning of the uncertainties of the emissivity windowing and Gaussian statistical combination techniques. While the imposed bounds may be correct—containing the proper values within their range—they remain agnostic to the underlying physical properties of metals that determine the true emissivity. They espe- cially disregard the powerful physical constraint imposed by emis- sivity at one wavelength upon emissivity at a neighboring wavelength. Total temperature calculated from a physically con- strained emissivity probability distribution can drive more precise emissivity estimates, prevent unphysical emissivity assumptions, and relieve pathological uncertainty bounds. FIG. 6. Spectral bands in experiment 2S-598. A 1550 nm notch in the longest wavelength channel blocks non-thermal light from fiber interferometry measurements. E. The Gaussian method for multiple wavelengths When temperatures calculated at each measured wavelength are in agreement, uncertainty may be bounded by the least high bound TH and the greatest low bound TL. When there is some disagreement between temperatures calculated at differing wavelengths, uncertainty may instead be bounded by the greatest high bound and least low bound. This technique makes no particu- lar assumption of the relative probability of any temperature between TL and TH. Temperature and uncertainty calculations for numerous shots are given in the paper of Jensen et al.15 The emis- sivity window method calculation for shot 2S-598 is plotted in a figure in that work as well. Repeating this calculation, using the shot 2S-598 data, produces T ¼ 1992 + 68 K, essentially identical to the published result. The calculated uncertainty is slightly The Gaussian method possesses some advantages and some drawbacks compared with the windowing method. It creates asym- metric temperature probability distributions, P(T), reflecting the decreased probability of lower temperatures, and increased proba- bility of higher temperatures, when calculating P(T) from a sym- metric P(ϵ) distribution. It also naturally avoids the non-physically small bounds that can be calculated by the window technique when measurements disagree. The Gaussian method temperature and its error bars are generally dominated by the measurement with the narrowest temperature probability distribution. For measurements with good inter-channel agreement, the Gaussian method over- weights those measurements at longer wavelengths or in spectral bands with greater emissivity uncertainty, under-constraining T. The magnitude and width of the Gaussian method temperature 131, 185901-6 131, 185901-6 J. Appl. Phys. 131, 185901 (2022); doi: 10.1063/5.0089457 © Author(s) 2022 J. Appl. Phys. 131, 185901 (2022); doi: 10.1063/5.0089457 © Author(s) 2022 Journal of Applied Physics scitation.org/journal/jap ARTICLE FIG. 5. Gaussian method temperature calculation for shot 2S-598. Top left: Gaussian P(ϵ) distribution used at all λ. Top center: T(ϵ) for Ch 1 L measure- ment. Top right: Probability of T, given L, P(ϵ). Bottom: Temperature probability for Ch 1–5 of the measurement, and multiplied total temperature probability, TGauss for all wavelengths. The mean of TGauss is 1967 K and the uncertainty bounds are 1923 and 2030 K. shapes than the narrow-band approximation. Next, Metropolis– Hastings simulations explicitly probe probability distributions of temperature and emissivity. Each refinement adds complexity and computational overhead, but the benefits include greater confidence and enhanced physical insight. IV. REFINED ANALYSIS Several refinements of pyrometry analysis are considered here. First, rigorous integration more accurately represents spectral J. Appl. Phys. 131, 185901 (2022); doi: 10.1063/5.0089457 © Author(s) 2022 J. Appl. Phys. 131, 185901 (2022); doi: 10.1063/5.0089457 © Author(s) 2022 J. Appl. Phys. 131, 185901 (2022); doi: 10.1063/5.0089457 131, 185901-7 131, 185901-7 © Author(s) 2022 Journal of Applied Physics scitation.org/journal/jap FIG. 7. Cerium temperature bounds for 0.1–1 emissivity for measured (blue) and perturbed (red, offset for visibility) band radiance. Maximum temperatures for ϵ ¼ 0:2 (circle) and ϵ ¼ 0:3 (triangle) are shown on the longest wavelength band. The dashed horizontal line is a weighted average of the five bands. FIG. 8. Temperatures calculated from Metropolis–Hastings sampling of three emissivity probability distributions. FIG. 7. Cerium temperature bounds for 0.1–1 emissivity for measured (blue) and perturbed (red, offset for visibility) band radiance. Maximum temperatures for ϵ ¼ 0:2 (circle) and ϵ ¼ 0:3 (triangle) are shown on the longest wavelength band. The dashed horizontal line is a weighted average of the five bands. FIG. 7. Cerium temperature bounds for 0.1–1 emissivity for measured (blue) and perturbed (red, offset for visibility) band radiance. Maximum temperatures for ϵ ¼ 0:2 (circle) and ϵ ¼ 0:3 (triangle) are shown on the longest wavelength band. The dashed horizontal line is a weighted average of the five bands. FIG. 8. Temperatures calculated from Metropolis–Hastings sampling of three emissivity probability distributions. FIG. 7. Cerium temperature bounds for 0.1–1 emissivity for measured (blue) and perturbed (red, offset for visibility) band radiance. Maximum temperatures for ϵ ¼ 0:2 (circle) and ϵ ¼ 0:3 (triangle) are shown on the longest wavelength band. The dashed horizontal line is a weighted average of the five bands. FIG. 8. Temperatures calculated from Metropolis–Hastings sampling of three emissivity probability distributions. are randomly rejected based on relative likelihood. The process is repeated 1106 times for each model. maximum temperature for ϵ ¼ 0:2–0.3, which are 600–900 K lower than the ϵ ¼ 0:1 bound. Gray body model simulations were performed with an emis- sivity range of 0.2–0.8. A histogram of temperature states for this model is shown in Fig. 8. The median temperature was 1978 K, and 90% of values fall within 1960–1995 K. Temperature results are nearly normal with a mean value of 1978 K and standard deviation of 11 K. Median emissivity was 0.506, with 90% of values within 0.481–0.533. B. Metropolis–Hastings simulations The Metropolis–Hastings algorithm44,45 was employed to stat- istically sample several emissivity models. The gray body model uses one adjustable parameter to describe emissivity at all wave- lengths. The gray band model uses five adjustable parameters to describe constant emissivity within each spectral band. The safe empirical model (Appendix B) uses two adjustable parameters to describe continuous emissivity variation across all ways; higher order versions are supported but not considered here. Safe emissivity simulations were drawn from 0:2 A 0:8 (λ0 ; 500 nm) and 2 b1 0. As noted above, only one of these parameters was drawn per iteration. The leftmost histogram in Fig. 8 shows temperature results from the simulation. The median temperature was 1926 K, with a 90% confidence region of 1899–1962 K. The mean temperature was 1928 K with a 19 K stan- dard deviation. Model parameters are drawn from a simplified beta distribu- tion Figure 9 shows median emissivity spectra for the gray band and safe model simulations. The former is consistent with the gray body result in almost every band, although the emissivity in the second longest band may be systemically higher. The safe model is systematically higher than the gray body model, especially at short wavelengths. tion dP dq ¼ 6 (q q1)(q2 q) (q2 q1)3 q1 q q2, 0 otherwise, 8 < : (13) (13) where parameter q is never below q1 or greater than q2. The optimal temperature is calculated for the radiances given in Table I using the emissivity for each parameter draw. After the first draw, parameter states that improve agreement between measured and calculated radiance are always kept. States that reduce agreement IV. REFINED ANALYSIS Very similar results were obtained for constant emis- sivity drawn from 0.1 to 1. The red bounds in Fig. 7 illustrate the effects of stray light in a hypothetical measurement perturbation. Suppose that all channels were contaminated by non-thermal light (Appendix A) equivalent to 1% of the power in the longest wavelength channel. Such con- tamination has the greatest impact on the short wavelength channel, offsetting the benefits of that channel’s tolerance of emis- sivity variation. Long wavelength channels may provide less tem- perature constraint than short wavelength channels for a given emissivity range, but those channels suffer lower systematic error from non-thermal light. Gray band model simulations were also drawn from the range 0.2–0.8, with no correlation between spectral bands. To maintain reasonable acceptance rates, only one emissivity was changed per iteration, randomly alternating between spectral bands. The broader, slightly skewed histogram of resulting temperatures is shown in Fig. 8. The median temperature was 1978 K, with a 90% confidence region of 1936–2040 K. The mean temperature was 1982 K with a 32 K standard deviation. 0 0 35 50 V. DISCUSSION Table III summarizes temperature results from Secs. III and IV. Consistency between methods and uncertainty trends are discussed J. Appl. Phys. 131, 185901 (2022); doi: 10.1063/5.0089457 © Author(s) 2022 J. Appl. Phys. 131, 185901 (2022); doi: 10.1063/5.0089457 131, 185901-8 © Author(s) 2022 Journal of Applied Physics ARTICLE scitation.org/journal/jap FIG. 9. Emissivity spectra for the gray band (above) and safe model (below) simulations. Red lines indicate the gray body result. measurement wavelengths produces large uncertainty values; however the true temperature value is likely to be contained within these uncertainties. For window method calculations with 0:1 ϵ 1, the true temperature almost certainly lies within uncer- tainty, but uncertainty is ≏10%. Better-informed emissivity bounds of 0:3 ϵ 0:8 for this particular experiment produced, in both spectrally integrated and narrow-band calculations, a T value that agrees with all other calculations within moderate error bars of ≏4%. Combining multiple wavelengths assuming Gaussian rather than square function emissivity probability calculated modestly lower T and uncertainty (≏3%). Both the simple window and Gaussian calculations agree with the refined analyses within uncertainty. Employing complete spectral band integration, performing Metropolis–Hastings sampling of the emissivity distribution and, finally, adding emissivity constraints anchored to the physical nature of emissivity in metals produced better constrained and pos- sibly more accurate T calculations. Metropolis–Hastings sampling of 0:2 ϵ 0:8 across all spectrally integrated bands inferred T nearly identical to the published result and the simple analyses shown here. However, apparent uncertainty is reduced to ≏1%, a value that seems too small. Repeating the calculation with emissiv- ity allowed to vary between bands again produces the same inferred T but with ≏3% uncertainty bounds. Applying a constraining model founded on the nature of emissivity in metals and sampling a range of physically plausible constraint parameters calculates T ≏2–3% lower, with ≏2% uncertainty. As discussed in Appendix A, dynamic spectral emissivity may be directly measured in some specially designed experiments. Hixson and La Lone (Hixson–La Lone)39 recently published a study measuring both spectral radiance and spectral reflectance on shocked Ce. The specifics of the experiment are described in that work. One experiment (13) measured radiance and reflectance at a Ce–LiF interface stress value of 21:7 + 0:1 GPa; we compare this with our analysis, where radiance was measured at an interface stress of 21:78 + 0:05 GPa. A. Temperature comparison A range of simple techniques to infer dynamic emissivity can be used with varying levels of deduced uncertainty. If radiance is measured with no emissivity knowledge, the absolute low bound radiance temperature can be computed with certainty. We find that this value is more than 10% too low despite the relatively high emis- sivity in our example experiment. Measuring at multiple wavelengths and assuming only constant emissivity completely bounds tempera- ture. However, the two-color estimates of temperature strongly dis- agree with one-another and over- or under-estimate T in various cases. Fitting five measured wavelengths, in the narrow-band approx- imation, to one blackbody curve computes a T that appears some- what high but agrees with the reported temperature and with most calculations. Placing absolute bounds on emissivity at several V. DISCUSSION Nominally, we would expect our result to be about 6 K higher due to the slight difference in shock pres- sure. Hixson–La Lone reports T ¼ 1912 + 38 K. This result agrees with all but one of our calculations but it is systematically lower in T by 1%–5%. 24 October 2024 04:35:50 FIG. 9. Emissivity spectra for the gray band (above) and safe model (below) simulations. Red lines indicate the gray body result. here. Emissivity spectra obtained from refined analysis are also com- pared to static and dynamic results reported elsewhere. Several temperature measurements in the Hixson–La Lone study were performed at Ce–LiF interface stresses similar to those in the Jensen et al. study. We used the radiance levels recorded by Jensen et al. to calculate interface temperature via the rigorous gray body, gray band, and safe models. We adjusted the calculated tem- peratures to account for the modest differences (0.06–0.50 GPa) in interface stress. On comparable experiments, the root-mean-square deviation reported in the two studies as-published is 8.7%. Applying the rigorous gray body and gray band models to the Jensen et al. data reduces this to 7.0% and 7.7%, respectively. The rigorous safe model deviation was 5.9%, a ≏30% reduction. B. Emissivity comparison Figure 10 compares emissivity results from Secs. I–IV to cal- culations based on the optical constants of cerium at ambient con- ditions.46 One calculation considers pure metal emitting in a host J. Appl. Phys. 131, 185901 (2022); doi: 10.1063/5.0089457 © Author(s) 2022 131, 185901-9 J. Appl. Phys. 131, 185901 (2022); doi: 10.1063/5.0089457 Journal of Applied Physics scitation.org/journal/jap TABLE III. Calculated temperatures for shot 2S-598. Temperature (K) Uncertainty (K) Band calculation Temperature (K) Uncertainty (K) Temperature (K) Uncertainty (K) Band calculation Radiance temperature ≥1704 … Narrow Two-color pyrometry, neighboring channels 1857–2510 ∼300 Narrow Free graybody fit 2026 ∼65 Narrow Window method with conservative ϵ = [0.1 −1.0] 2115 +125, −215 Integrated Window method reported in original work 1993 84 Integrated Window method calculated in this work 1992 68 Narrow Gaussian method 1967 +63, −44 Narrow Rigorous gray body 1978 22 Integrated Rigorous gray band 1982 64 Integrated Rigorous safe model 1928 38 Integrated Hixson–La Lone ϵ measurement applied to 2S-598 2064 … Integrated Hixson–La Lone reported Ta with ϵ measurement 1912 38 Narrow aExperiment “13,” at lower stress, with shock T ∼6 K lower. FIG. 10. Comparison of simulated emissivity with ambient cerium46 emitting into vacuum and an oxide layer (nh ¼ 1:8–2.2), as well as measured ambient and dynamic emissivity of a Ce target.39 of cerium oxide is around 2,47 so the value of 1.8–2.2 are used to generate a plausible emissivity band. Measurements of an assembled Ce–LiF target measured by Hixson–La Lone are also shown. The ambient emissivity is measured across the spectrum, while dynamic emissivity is reported at two NIR wavelengths. The authors have pro- vided us with their data for that experiment. Applying these values we calculate a temperature of 2065 K on band 1 and 2062 K on band 2 of the 2S-598 radiance data. While the analysis presented in both previous studies and the analysis presented here generally agree within uncertainty on dynamic temperature, applying dynamic emissivity measured in one experiment to radiance data measured on a separate experi- ment produces disagreement. This must be due to differences in the precise surface finish, surface oxidation state, and other details of exact target finish. The Hixson–La Lone team developed a method of sample work under inert atmosphere to minimize surface oxidation. B. Emissivity comparison The group that published the experiment ana- lyzed in this work conducted one experiment on a sample using the same handling method; the results of this shot were in agree- ment with the Hixson–La Lone team’s measurements at nearly the same pressure.39 The best currently demonstrated method to account for surface oxide may be development of careful target fin- ishing, handling, and assembly under inert atmosphere, to mini- mize the effect of surface oxidation. Even so, dynamic emissivity measurements may vary from shot to shot even for nominally iden- tical target and experiment designs. DATA AVAILABILITY The data that support the findings of this study are available from the corresponding authors upon reasonable request. p g y q To further advance the capabilities of dynamic temperature measurement, we suggest two areas of focus. Radiance measure- ments in most cases necessarily measure at an interface between the emitter and another material. In some experiments, complex interface phenomena cannot be eliminated from the design. Dedicated experiments are needed that directly assess the magni- tude of the uncertainties discussed above in these geometries. Examples of this work include measurement of material effusivity49 and LiF thermal conductivity under shock compression. Focus should also be applied to new experiment designs that can elimi- nate or work around such effects. Thermal interfaces that directly express or quickly equilibrate to the probed state eliminate the uncertainty of inferring temperature away from the interface.50 Measurement of thermal equilibration across the measurement interface bounds the initial temperature profile across an interface.18 1. Measurement wavelengths Direct measurements of dynamic spectral emissivity are the most physically certain method to calculate temperature from radiometric data. However, in general, the emissivity measured in one experiment cannot generally be used to calculate temperature from radiance collected in another experiment. This may be possi- ble for experiments with pure metals, such as those coated onto optical windows and capped with a passivating layer, or metals with very little surface chemistry, glued to windows with identical glue bonds. Calculations of emissivity, beginning with measured ambient target emissivity, forced to vary in a physically plausible manner, can reproduce measured values closely enough to shift the source of greatest T uncertainty from radiance analysis to the nature of the measured radiant interface. Equations (4) and (5) establish that calculated temperature precision at a steady measurement uncertainty is increased by mea- surement at the shortest feasible wavelengths. However, radiance emission at wavelengths smaller than the peak of the Planck func- tion decreases dramatically with decreasing wavelength. At some wavelength, any non-zero background will become larger than thermal emission. In this case, an attempt to measure at the small- est practical wavelengths can be combined with a “go bluer” analy- sis technique: temperature analysis begins at the largest wavelength and proceeds to shorter wavelengths via one of the techniques described in Sec. III. Analysis is then halted at the first wavelength where radiance values are sufficiently close to background levels to produce significant disagreement with the more reliable longer wavelength measurements. All smaller wavelength data are rejected. A mathematical solution for the smallest feasible measurement wavelength, λmin, as a function of T is not readily expressible. However, λmin can be calculated explicitly for real optical collection and detection system as a function of temperature and detector sensitivity. APPENDIX A: RADIANCE MEASUREMENT CONSIDERATIONS Before the analysis of temperature can begin, measurement wavelengths must be carefully chosen, and pure blackbody radiance must be assured at each included wavelength. In practice, this means that there must be a single radiation temperature and no background (non-thermal) radiation contaminating the measure- ment. If this condition can be achieved, dynamic spectral emissivity variation is the single largest source of uncertainty for temperature calculation. In some cases, direct experimental tests of dynamic emissivity can be made. The authors have no conflicts to disclose. The authors have no conflicts to disclose. VI. CONCLUSIONS AND FUTURE WORK FIG. 10. Comparison of simulated emissivity with ambient cerium46 emitting into vacuum and an oxide layer (nh ¼ 1:8–2.2), as well as measured ambient and dynamic emissivity of a Ce target.39 In experiments where emissivity cannot be measured, a range of simple radiance analysis techniques produce T estimates with uncertainties of ≏5% and possibly as good as ≏3%–4% when applied to excellent data. These calculations generally agree with one another at the ≏5% level. We find that statistical analyses, especially when constrained by the physical nature of ϵ(λ), can allow T uncertainty in radiance-only experiments to approach the index of 1, while the other calculations are based on larger host indices. The latter occurs almost instantly when cerium is exposed to air, rapidly oxidizing the surface. The characteristic refractive index J. Appl. Phys. 131, 185901 (2022); doi: 10.1063/5.0089457 © Author(s) 2022 J. Appl. Phys. 131, 185901 (2022); doi: 10.1063/5.0089457 131, 185901-10 Journal of Applied Physics ARTICLE scitation.org/journal/jap multi-mission laboratory managed and operated by National Technology and Engineering Solutions of Sandia LLC, a wholly owned subsidiary of Honeywell International, Inc., for the U.S. Department of Energy’s National Nuclear Security Administration under Contract No. DE-NA0003525. This paper describes objective technical results and analysis. Any subjective views or opinions that might be expressed in the paper do not necessarily represent the views of the U.S. Department of Energy or the United States Government. ≏2% uncertainty possible with emissivity measurement in some cases. The “Rigorous Safe Model” appears to be the best of these methods, agreeing with small but plausible uncertainty with inde- pendent measurements made on nearly identical experiments with different material surface conditions, with and without emissivity measurement. Several fundamental uncertainties limit the inference of bulk state temperature from the measured interface temperature to a similar ≏2%–5% level of uncertainty:14,48,49 shock temperatures of the window and window-sample attachment bond layer; thermal properties of samples, optical windows, and attachment layers or films; bond layer and shock front thickness; loading trajectories across mechanical interfaces at or near the measurement. Pyrometric inference of dynamic temperature in dedicated shock experiments likely now carries error bars similar to these other experimental uncertainty sources. For most experiments, under- standing systematic errors in these sources is of similar or greater importance to honing radiance analysis techniques. 3. Dynamic spectral emissivity ratio method If an experimental geometry can be implemented to eliminate non-thermal light in the FOV, uncertainty in spectral emissivity is the dominant factor in temperature uncertainty. A direct measure- ment of absolute spectral emissivity, when possible, increases preci- sion of temperature measurement. Implementing this method under dynamic loading can be very challenging. A fast laser polar- imeter can possibly be used29 but suffers from several drawbacks. It is highly sensitive to dynamic tilt or bow in any sample, to anvil window optical birefringence in supported shock experiments, and to details of specularity and non-Lambertian diffuse reflection. FIG. 11. Accessible measurement wavelength contours for various tempera- tures (ϵ ¼ 1). Wavelength selection is guided by selecting the desired measure- ment temperature and then moving vertically until intersecting the appropriate sensitivity contour. Sensitivity here is expressed as 1 mV measured signal on a detector in an experiment collecting light from an area of 1 mm2, into a numeri- cal aperture of 0.20, through an optical bandwidth of 0.1 μm (100 nm). can be readily scaled for collection area, optical bandwidth, and numerical aperture for a particular experiment; they are meant as a rough guide. p y Nevertheless, a method to measure the ratio of dynamic reflec- tance to static reflectance in certain experiments has been success- fully developed.9,30,54 An integrating sphere illuminates the sample with broadband light from nearly 2π overhead steradians while an optical probe measures the ratio of the time-resolved normal reflec- tance signal over time to the normal reflectance signal before shock breakout. This links the dynamic spectral reflectance to the ambient spectral reflectance, which can be readily measured under static conditions with proper optical geometry.22 Kirchoff’s law of thermal radiation converts static and dynamic reflectance to static and dynamic emissivity. Unfortunately, the integrating sphere design required to measure the dynamic spectral reflectivity is limited to use in certain experimental geometries. The sphere must be directly attached to the anvil window or other measurement surface, making it highly impractical for measurements of free- surfaces, surfaces with large or non-uniform curvature, converging or non-uniform shock waves, and other exotic geometries. The geometry of an illuminating sphere limits the minimum sphere size, the maximum anvil window thickness, and the target dimen- sions. Additionally, the sphere generally requires a relatively specu- lar surface that does not vary drastically in specularity throughout the experiment. 3. Dynamic spectral emissivity ratio method Generally, this is acceptable in temperature-focused experiments that can be designed around these requirements; it can be challenging or impossible to add spheres to assemblies with complex shapes or irregular surface finishes, or to very small A more conservative temperature measurement will collect radiance at wavelengths near the peak of the Planck emission curve for a given temperature. This is a robust measurement, assuring the greatest possible signal levels. However, calculated temperature pre- cision from a given radiance or emissivity uncertainty can be improved by adding measurements at shorter wavelengths if there is sufficient flux. There is no practical benefit to measuring at wave- lengths larger than the peak of the Planck curve, where signal is less and calculated uncertainty is greater. In general, measurement wavelengths are best chosen to lie between the blackbody emission peak and the smallest wavelength with measurable signal. ACKNOWLEDGMENTS We would like to thank Lynn Veeser, Brandon La Lone, and Jerry Stevens for providing us their data and for their discussion, criticism, and insights regarding the analyses presented in this work. Los Alamos National Laboratory is operated by Triad National Security, LLC, for the National Nuclear Security Administration of U.S. Department of Energy under Contract No. 89233218CNA000001. Sandia National Laboratories is a Figure 11 shows the nature of the sensitivity curves for an optical collection scheme common for a typical shock experiment. Light collection involves a sample area of 1 mm2, a numerical aper- ture of 0.2, and an optical bandwidth of 100 nm. These calculations J. Appl. Phys. 131, 185901 (2022); doi: 10.1063/5.0089457 © Author(s) 2022 131, 185901-11 131, 185901-11 J. Appl. Phys. 131, 185901 (2022); doi: 10.1063/5.0089457 © Author(s) 2022 Journal of Applied Physics ARTICLE scitation.org/journal/jap FIG. 11. Accessible measurement wavelength contours for various tempera- tures (ϵ ¼ 1). Wavelength selection is guided by selecting the desired measure- ment temperature and then moving vertically until intersecting the appropriate sensitivity contour. Sensitivity here is expressed as 1 mV measured signal on a detector in an experiment collecting light from an area of 1 mm2, into a numeri- cal aperture of 0.20, through an optical bandwidth of 0.1 μm (100 nm). period. The field of view (FOV) must be optically defined to miti- gate light leakage from sources such as light from high explosive (HE) burn or flyer impact flash specific to the shock wave drive configuration of the experiment. Spectral filtering is required to eliminate collection of injected probe light such as that from optical velocimetry. Transmitting fiber runs must be designed to shield direct optical and radiation-induced backgrounds. Spectral and temporal resolution enable some simple tech- niques to mitigate the presence of certain non-thermal light sources in temperature analysis. Careful modeling of wave propaga- tion can indicate the timing of transient light sources from impact flash, certain shock releases, optical window edge-release, or bond layer compression, to enable their removal by gating. A carefully constrained “null” measurement of only the optical path can be used to subtract backgrounds that do not originate from the target. In many cases, background sources are known to possess certain spectral characteristics.53 Measurements at these wavelengths may be excluded. REFERENCES 1G. Duvall and R. Graham, “Phase transitions under shock wave loading,” Rev. Mod. Phys. 49, 523 (1977). dϵ dλ ¼ ϵ(λ) A 1 ϵ(λ) 1 A g(λ): (B1) (B1) 2W. Nellis, “Dynamic compression of materials: Metallization of fluid hydrogen at high pressures,” Rep. Prog. Phys. 69, 1479 (2006). Here, ϵ(λ0) ; A for an arbitrarily reference wavelength λ. The slope function g(λ) controls how emissivity varies away from the reference value. 3W. J. Carter, J. Fritz, S. Marsh, and R. McQueen, “Hugoniot equation of state of the lanthanides,” J. Phys. Chem. Solids 36, 741–752 (1975). 4 4S. Kormer, M. Sinitsyn, G. Kirillov, and V. Urlin, “Experimental determination of temperature in shock-compressed nacl,” Sov. Phys. JETP 21, 689 (1965). 5 Integrating Eq. (B1) yields an intrinsically safe model 5G. Lyzenga and T. J. Ahrens, “Multiwavelength optical pyrometer for shock ϵ(λ) ¼ A Aþ(1A)eh(λ) , h(λ) ; ðλ λ0 g(u) du: (B2) compression experiments,” Rev. Sci. Instrum. 50, 1421–1424 (1979). 6 6W. Nellis, F. H. Ree, R. Trainor, A. Mitchell, and M. Boslough, “Equation of (B2) state and optical luminosity of benzene, polybutene, and polyethylene shocked to 210 GPa (2.1 Mbar),” J. Chem. Phys. 80, 2789–2799 (1984). 24 Oct y 7C. Yoo, N. Holmes, M. Ross, D. Webb, and C. Pike, “Shock temperatures and ctobe 7C. Yoo, N. Holmes, M. Ross, D. Webb, and C. Pike, “Shock temperatures and melting of iron at Earth core conditions ” Phys Rev Lett 70 3931 (1993) tober melting of iron at Earth core conditions,” Phys. Rev. Lett. 70, 3931 (1993). 8 er 20 Large positive values of h lead to ϵ ! 1, while large negative values lead to ϵ ! 0. No matter what function form is chosen for g or h, the emissivity function is never unphysical. 8D. Partouche-Sebban, D. Holtkamp, J. Pélissier, J. Taboury, and A. Rouyer, “An 024 investigation of shock induced temperature rise and melting of bismuth using high-speed optical pyrometry,” Shock Waves 11, 385–392 (2002). 9 04:35:5 high-speed optical pyrometry,” Shock Waves 11, 385–392 (2002). 35:5 9B. La Lone, P. 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Experiments utilizing transparent anvil windows to support the shocked surface must be designed to avoid optical backgrounds from bulk emission of the window material (e.g., sapphire51,52) and release at the edges.53 The bond between materials must remain transparent through the measurement J. Appl. Phys. 131, 185901 (2022); doi: 10.1063/5.0089457 © Author(s) 2022 131, 185901-12 J. Appl. Phys. 131, 185901 (2022); doi: 10.1063/5.0089457 Journal of Applied Physics ARTICLE scitation.org/journal/jap The trivial case g(λ) ¼ 0 leads to h(λ) ¼ 0, which is simply the gray body model. Consider the series expansion (approximately, ,1 cm target diameter or ,5 cm diagnostic access area) experiments. Recent studies have used the integrating sphere reflectance technique to measure change in spectral emissivity under dynamic loading in several metals at various shock pressures.9,39,55 The greatest factor driving change in dynamic emissivity of metals seems to be phase change. However, the nature of dynamic emis- sivity variation appears to depend on a large number of additional factors. These include: wavelength, shock pressure, specific metal, nature of the surface oxide, and thickness of any window-sample bond layer. At this time, more generalized rules or guiding princi- ples of dynamic emissivity trends cannot be given in most cases. h(λ) ¼ X M m¼1 bm λ λ0 λ0 m , (B3) (B3) where b1 is emissivity slope at the reference wavelength. Figure 12 shows several examples for values of b1 , 0 that qualitatively repre- sent metal emissivity. More detailed structure can be represented with higher-order terms. This approach is similar to the logistic curve discussed in Ref. 56, excluding unphysical cases where metals could emit more strongly at long wavelengths than short wavelengths. REFERENCES Siroux, and B. Desmet, “Measurements of brake disc surface temperature and emissivity by two-color pyrometry,” Appl. Therm. 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Jensen et al., “Measurement of the shock-heated melt curve of lead using pyrometry and reflectometry,” J. Appl. Phys. 97, 043521 (2005). 35Y. A. Levendis, K. R. Estrada, and H. C. REFERENCES Mitchell, “The temperature of shock-compressed water,” J. Chem. Phys. 76, 6282–6286 (1982). 11 11S.-N. Luo, J. A. Akins, T. J. Ahrens, and P. D. Asimow, “Shock-compressed MgSiO3 glass, enstatite, olivine, and quartz: Optical emission, temperatures, and melting,” J. Geophys. Res.: Solid Earth 109, 05205, https://doi.org/10.1029/ 2003JB002860 (2004). 12J. D. Bass, B. Svendsen, and T. J. Ahrens, “The temperature of shock com- pressed iron,” High Pressure Res. Min. Phys. 39, 393–402 (1987). 13 13W. Nellis and C. Yoo, “Issues concerning shock temperature measurements of iron and other metals,” J. Geophys. Res. 95, 21749–21752, https://doi.org/ 10.1029/JB095iB13p21749 (1990). 14T. Hartsfield, B. La Lone, G. Stevens, L. Veeser, and D. Dolan, “Thermal inter- faces in dynamic compression experiments,” J. 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Veeser, “Ejecta transport, breakup and conversion,” J. Dyn. Behav. Mater. 3, 334–345 (2017). 17A. Seifter and A. W. Obst, “About the proper wavelength for pyrometry on shock physics experiments,” Int. J. Thermophys. 28, 934–946 (2007). J. Appl. Phys. 131, 185901 (2022); doi: 10.1063/5.0089457 131, 185901-13 131, 185901-13 © Author(s) 2022 Journal of Applied Physics ARTICLE scitation.org/journal/jap 37B. Müller and U. Renz, “Development of a fast fiber-optic two-color pyrometer for the temperature measurement of surfaces with varying emissivities,” Rev. Sci. Instrum. 72, 3366–3374 (2001). 18T. M. Hartsfield et al., “Thermal interfaces in dynamic compression experi- ments,” J. Appl. Phys. 128, 015903 (2022). pp y 19E. Tiesinga, P. J. Mohr, D. B. Newell, and B. N. Taylor, see https://physics.nist. 38J. Thevenet, M. REFERENCES Chem. Phys. 21, 1087–1092 (1953). 26K. Boboridis, A. Seifter, A. Obst, and D. 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https://openalex.org/W4378211686	https://oem.bmj.com/content/oemed/early/2023/05/24/oemed-2022-108601.full.pdf	English	null	Testicular germ cell tumour risk by occupation and industry: a French case–control study – TESTIS	Occupational and environmental medicine	2,023	cc-by	12,430	WHAT THIS STUDY ADDS ⇒Consistent with previous studies, an increased risk of TGCT among agricultural, electrical and electronics, and salesmen workers was observed. g g Results A positive association was observed between TGCT and occupation as agricultural, animal husbandry worker (ISCO: 6–2; OR 1.71; 95% CI (1.02 to 2.82)), as well as salesman (ISCO: 4–51; OR 1.84; 95% CI (1.20 to 2.82)). An increased risk was further observed among electrical fitters and related, electrical and electronics workers employed for 2 years or more (ISCO: 8–5; OR≥2 g g Results A positive association was observed between TGCT and occupation as agricultural, animal husbandry worker (ISCO: 6–2; OR 1.71; 95% CI (1.02 to 2.82)), as well as salesman (ISCO: 4–51; OR 1.84; 95% CI (1.20 to 2.82)). An increased risk was further observed among electrical fitters and related, electrical and electronics workers employed for 2 years or more (ISCO: 8–5; OR≥2 ⇒This observation suggests consistent associations over time of occupations with the risk of TGCT. on October 23, 2024 by guest. Protected by copyright. http://oem.bmj.com/ m to 2.82)). An increased risk was further observed among electrical fitters and related, electrical and electronics workers employed for 2 years or more (ISCO: 8–5; OR≥2 Original research ►Additional supplemental material is published online only. To view, please visit the journal online (http://dx.doi. org/10.1136/oemed-2022- 108601). ABSTRACT Obj i T ABSTRACT Objective Testicular germ cell tumours (TGCT) are the most common cancer in men of working age and its incidence has increased notably over the past 40 years. Several occupations have been identified as potentially associated with TGCT risk. The aim of this study was to further explore the relationship between occupations, industries and TGCT risk in men aged 18–45 years. Methods The TESTIS study is a multicenter case– control study conducted between January 2015 and April 2018 in 20 of 23 university hospital centers in metropolitan France. A total of 454 TGCT cases and 670 controls were included. Full job histories were collected. Occupations were coded according to the International Standard Classification of Occupation 1968 version (ISCO-1968) and industry according to the 1999 version of Nomenclature d’Activités Française (NAF-1999). For each job held, ORs and 95% CIs were estimated using conditional logistic regression. WHAT IS ALREADY KNOWN ON THIS TOPIC ⇒Current evidence has not allowed to identify consistent occupational risk factors for testicular germ cell tumours (TGCT). ⇒Current evidence has not allowed to identify consistent occupational risk factors for testicular germ cell tumours (TGCT). ⇒In the past, several occupations and industries have been suggested to increase the risk of TGCT, including agricultural workers, firemen, policemen, military personnel and industrial workers (paper, electric, plastic or metal). ⇒In the past, several occupations and industries have been suggested to increase the risk of TGCT, including agricultural workers, firemen, policemen, military personnel and industrial workers (paper, electric, plastic or metal). For numbered affiliations see end of article. Correspondence to Margot Guth, UMRESTTE, Université Claude Bernard Lyon 1, Lyon, 9405, France; margot.guth@univ-lyon1.fr ⇒Although TGCT incidence has increased further, only few studies have been published in recent years, while occupational health regulations may have lowered workplace exposures. ⇒Although TGCT incidence has increased further, only few studies have been published in recent years, while occupational health regulations may have lowered workplace exposures. Received 10 August 2022 Accepted 4 May 2023 HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY ⇒The findings of this study offer important insights into the possible role of some high- risk occupations in the development of TGCT, suggesting the need to reinforce work health and safety measures. ⇒The findings of this study offer important insights into the possible role of some high- risk occupations in the development of TGCT, suggesting the need to reinforce work health and safety measures. p y y ≥2 years 1.83; 95% CI (1.01 to 3.32)). Analyses by industry supported these findings. Conclusions Our findings suggest that agricultural, electrical and electronics workers, and salesmen workers experience an increased risk of TGCT. Further research is needed to identify the agents or chemicals in these high-risk occupations which are relevant in the TGCT development. y ⇒Further studies are needed to investigate the agents involved in the high-risk occupations identified in our study and assess their association with the risk of TGCT. er 23, 2024 by guest. Protected by copyright. Trial registration number NCT02109926. Trial registration number NCT02109926. Trial registration number NCT02109926. Genetic predisposition, personal and family history of TGCT are established as risk factors for TGCT.2 Differing incidence rates between population groups and geographical regions,4 and evolving incidence in migrant populations5 suggest a multifactorial origin, with complex interactions between genetic predisposition and yet unknown environmental factors.3 No specific environmental exposures have yet been identified, but endocrine disrupting chemicals (EDCs) are strongly suspected to play a role in TGCT development, and have been guest. Protected by copyright. Testicular germ cell tumour risk by occupation and industry: a French case–control study – TESTIS Margot Guth ‍ ‍ ,1 Astrid Coste,2,3 Marie Lefevre,1 Floriane Deygas,1 Aurélie Danjou,4 Shukrullah Ahmadi,4 Brigitte Dananché,3 Olivia Pérol,3 Helen Boyle,5 Joachim Schüz,4 Louis Bujan,6,7 Catherine Metzler-Guillemain,7,8 Sandrine Giscard d’Estaing,7,9 Marius Teletin,7,10 Berengere Ducrocq,7,11 Cynthia Frapsauce,7,12 Ann Olsson ‍ ‍ ,4 Barbara Charbotel,1,13 Béatrice Fervers,2,3 and the TESTIS study group ►Additional supplemental material is published online only. To view, please visit the journal online (http://dx.doi. org/10.1136/oemed-2022- 108601). on October 23, 2024 by guest. Protected by copyrigh http://oem.bmj.com/ Occup Environ Med: first published as 10.1136/oemed-2022-108601 on 25 May 2023. Downloaded from Workplace on October 23, 2024 by guest. Prote http://oem.bmj.com/ Occup Environ Med: first published as 10.1136/oemed-2022-108601 on 25 May 2023. Downloaded from Study design The TESTIS study is a multicenter case–control study conducted between 2015 and 2018, in 20 out of the 23 university hospital (UH) in metropolitan France.17 TESTIS included patients (aged 18–45 years) with histologically confirmed TGCT, referred to a Centre d'étude et de conservation d'œufs et de sperme humain (CECOS) for pretreatment sperm cryopreservation. Recruit- ment with rapid ascertainment of cases through the CECOS was the most promising design resulting from the pilot study, to increase participation rates.18 Two control groups with no personal history of testicular cancer or cryptorchidism were also recruited: group A—sperm donors and partners of women consulting for fertility disorders, with normal sperm production, in CECOS ; group B—partners of women treated for a patho- logical pregnancy in specialised maternity clinics, adjacent to the CECOS. TGCT cases were individually matched to a control A and a control B on the recruiting UH and year of birth (±3 years).17 The target population (ie, young adult men) is partic- ularly associated with low response rates,19 20 making it diffi- cult to obtain an unbiased random sample of general population controls.17 In absence of a perfect control group, we chose two control groups presenting different aspects of the general popu- lation, as suggested by Stang et al.21 Moreover, according to the ‘testicular dysgenesis syndrome’ hypothesis, TGCT, cryptorchi- dism, hypospadias and some fertility disorders are presumed to originate from common dysfunction in intrauterine genital development.4 By selecting controls assumed to be fertile (group B) or with normal sperm count (group A), and without history of cryptorchidism, the risk of recruiting controls with minor forms of this syndrome was reduced. Regional catchment populations INTRODUCTION l To cite: Guth M, Coste A, Lefevre M, et al. Occup Environ Med Epub ahead of print: [please include Day Month Year]. doi:10.1136/ oemed-2022-108601 © World Health Organization 2023. Licensee BMJ. Testicular germ cell tumours (TGCT) are a rare cancer, but the most common cancer affecting men of working age (15–44 years) with an increasing incidence over the past 40 years, particularly among Caucasian populations.1 2 TGCT account for approximately 98% of malignant testicular tumours in France and are classified into two main histological subtypes, namely seminoma and non-seminoma.3 Guth M, et al. Occup Environ Med 2023;0:1–11. doi:10.1136/oemed-2022-108601 1 Workplace on October 23, 2024 by guest. Protected by copyrigh http://oem.bmj.com/ Occup Environ Med: first published as 10.1136/oemed-2022-108601 on 25 May 2023. Downloaded from were similar for cases and both control groups.17 Participants provided written informed consent prior to study entry. were similar for cases and both control groups.17 Participants provided written informed consent prior to study entry. associated with increased incidence.3 6 Some organochlorines pesticides as well as polychlorinated biphenyls, hexavalent chro- mium and perfluorooctanoic acid (PFOA) have been associated with TGCT.7–11 Ascertainment of GCNIS-related TGCT cases Ascertainment of GCNIS-related TGCT cases has been detailed elsewhere.17 Briefly, cases were classified into seminoma and non- seminoma by a TGCT expert (HB). Given the low proportion of false-positive TGCT (5.1%), TGCT with missing pathology report (N=43) were included as cases. Therefore, we report the results of a French nationwide case– control study that investigated whether participants’ occupa- tional history, by occupation and industries, may increase TGCT risk, overall and stratified by histological subtype. This study focuses specifically on TGCT occurring in young men, derived from germ cell neoplasia in situ (GCNIS) according to the 2016 WHO classification.16 Research hypotheses are distinct from that of non-GCNIS-derived tumours.16 Coding of job titles h b h For each job, the occupation was coded according to the Inter- national Standard Classification of Occupation 1968 version (ISCO-1968) and industry according to the Nomenclature d’ac- tivités française 1999 version (NAF-1999). All coding was based on participants’ job title and task descriptions, blind to their case– control status. Some codes did not have the maximum number of digits (5 for ISCO, 4 for NAF) due to lack of information reported and were considered as aggregate codes. Aggregated codes were used in the code analyses up to their last known digit level and considered as missing for other levels. er 23, 2024 by guest. Protected by copyright. Study population Overall, 1463 eligible subjects were invited to participate in the study, and 1367 (93.4%) agreed to participate. The population structure and reasons for non-participation were detailed previ- ously.17 Of the 550 recruited cases, 96 were excluded: 21 non- GCNIS TGCT, 4 tumours not confirmed by pathology reports, 4 time from diagnosis to study inclusion >12 months, 1 missing date of diagnosis and 66 incomplete interviews (online supple- mental figure S1). Of the controls, 9 not born in ‘metropolitan France’ (ie, excludes French subjects born in the French over- seas territories), 5 with personal history of cryptorchidism and 133 with an incomplete interview were excluded. Finally, 1124 participants were included in the analyses. Data collection Exposure to EDCs or carcinogens can occur in the work- place and the careful study of related occupations could lead to new hypotheses. The literature suggests a higher risk in certain occupations, such as agricultural, metal, electrical, construction, firemen, salesmen and clerical workers.2 7 12 However, most studies were published more than 10 years ago.7 12 Recently, the International Agency for Research on Cancer classified occupa- tional exposure as a firefighter as ‘carcinogenic to humans’, with ‘limited’ evidence for testicular cancers.13 Occupational health regulations and preventive measures over the last decades may have changed some occupational exposures. For example, a decrease in the prevalence of French workers exposed to at least one carcinogenic, mutagenic and reprotoxic agents was observed from 13.7% to 10.6% in 2003–2010, in a context of tightened European regulations.14 Nevertheless, TGCT in Europe will further increase, particularly in historically low-risk countries.15 It is thus relevant to obtain current data to examine potential evolutions of occupation-TGCT associations over time to guide future interventions. Of the 1323 participants included and having agreed to partic- ipate, 1124 subjects were interviewed by trained interviewers (IPSOS Company) blinded to the participants’ case–control status. The telephone interview used a structured question- naire (85% response rate; cases: 87%; group A: 87%; group B controls: 79%). A detailed lifetime work history was recorded of all jobs and internships (>6 months): job title, industry, primary and secondary tasks performed, jobs’ start and end dates, compagnies’ name, address and activity, number of employees, and percentage of time worked. Participants received a docu- ment to prepare information on job history prior to the phone interview.17 Covariates d h Based on the literature, covariates established or suspected to be risk factors for TGCT22 23 were considered for adjustment (presented in table 1). These included perinatal factors: birth weight, gestational age, birth order, sibship size and born from multiple pregnancy. Further covariates included family history of testicular cancer; family history of cryptorchidism; personal tobacco smoking; cannabis use;24 and personal consumption of alcoholic beverages.25 We considered cannabis use and frequency of use in two periods: adolescence (12–17 years) and young adulthood (18–25 years), puberty being possibly considered as the key point of exposure.24 We used age at voice change as a proxy of timing of pubertal development to consider delayed y guest. Protected by copyright. Guth M, et al. Occup Environ Med 2023;0:1–11. Covariates d h doi:10.1136/oemed-2022-108601 2 Workplace Workplace Table 1 Characteristics of TGCT cases and controls (group A and group B), TESTIS study, N=1124 Characteristics TGCT cases (n=454) Group A controls (n=384) Group B controls (n=286) P value cases/all controls* P value controls A/controls B* n (%) n (%) n (%) Age at diagnosis (cas)/inclusion (controls) (years) <0.001 0.69 ≤25 64 (14.1) 20 (5.2) 12 (4.2) 26–30 106 (23.4) 80 (20.8) 64 (22.4) 31–35 113 (24.9) 131 (34.1) 108 (37.8) 36–40 85 (18.7) 95 (24.7) 67 (23.4) ≥41 43 (9.5) 58 (15.1) 35 (12.2) Missing 43 (9.5) 0 (0.0) 0 (0.0) Year of birth <0.001 0.15 <1975 37 (8.2) 33 (8.6) 17 (5.9) 1975–1979 80 (17.6) 82 (21.4) 46 (16.1) 1980–1984 117 (25.8) 119 (31.0) 99 (34.6) 1985–1989 130 (28.6) 105 (27.3) 98 (34.3) 1990–1994 70 (15.4) 40 (10.4) 23 (8.0) 1995–1999 20 (4.4) 5 (1.3) 3 (1.1) Education 0.10 0.002 Secondary 180 (39.7) 137 (35.7) 66 (23.1) 1–2 years university degree 97 (21.4) 91 (23.7) 57 (19.9) > 3 years university degree 128 (28.2) 113 (29.4) 121 (42.3) Other 48 (10.6) 43 (11.2) 42 (14.7) Missing 1 (0.2) 0 (0.0) 0 (0.0) Smoking status 0.12 0.10 Former smoker 102 (22.5) 99 (25.8) 72 (25.2) Current smoker 147 (32.4) 116 (30.2) 69 (24.1) Never smoker 205 (45.2) 169 (44) 145 (50.7) Birth weight (g) 0.49 0.99 <2500 25 (5.5) 20 (5.2) 13 (4.6) 2500–4000 356 (78.4) 318 (82.8) 243 (85) ≥4000 46 (10.1) 32 (8.3) 26 (9.1) Missing 27 (6.0) 14 (3.7) 4 (1.4) Gestational age (weeks) 0.16 0.25 ≤36 32 (7.1) 23 (6.0) 11 (3.9) >36 415 (91.4) 358 (93.2) 271 (94.8) Missing 7 (1.5) 3 (0.8) 4 (1.4) Born from multiple pregnancy 0.02 0.59 No 433 (95.4) 375 (97.7) 277 (96.9) Yes 21 (4.6) 8 (2.1) 9 (3.2) Missing 0 (0.0) 1 (0.3) 0 (0.0) Birth order 0.04 0.39 First 213 (46.9) 168 (43.8) 137 (47.9) Second 163 (35.9) 135 (35.2) 89 (31.1) Third 63 (13.9) 51 (13.3) 42 (14.7) Fourth and more 15 (3.3) 30 (7.8) 18 (6.3) Sibship size 0.03 0.70 1 25 (5.5) 36 (9.4) 23 (8.0) 2 192 (42.3) 139 (36.2) 115 (40.2) 3 153 (33.7) 116 (30.2) 90 (31.5) ≥4 84 (18.5) 92 (24) 58 (20.3) Missing 0 (0.0) 1 (0.3) 0 (0.0) Personal history of inguinal hernia 0.01 0.20 No 414 (91.2) 362 (94.3) 275 (96.2) Yes 39 (8.6) 22 (5.7) 10 (3.5) Missing 1 (0.2) 0 (0.0) 1 (0.4) Personnal history of testicular trauma 0.004 0.53 No 387 (85.2) 351 (91.4) 260 (90.9) Yes 67 (14.8) 33 (8.6) 26 (9.1) continued on October 23, 2024 by guest. on October 23, 2024 by guest http://oem.bmj.com/ Occup Environ Med: first published as 10.1136/oemed-2022-108601 on 25 May 2023. Downloaded from on October 23, 2024 by guest. Protected by copyright. http://oem.bmj.com/ published as 10.1136/oemed-2022-108601 on 25 May 2023. Downloaded from puberty, a protective factor for TGCT.26 Although biologically questionable, TGCT risk has been reported in several studies to be associated with a personal history of testicular trauma.23 The selection of covariates followed a two-step process: first, we identified variables associated with TGCT in univariate anal- yses and selected those with p<0.20. All selected covariates were then included in one single regression model, and a manual backward stepwise selection procedure was performed. The final model included the following variables significantly associated with TGCT (p<0.05): sibship size, being born from multiple pregnancy, personal history of testicular trauma, family history of TGCT, and family history of cryptorchidism. We used the same adjusted model for all analyses. was assessed as an ordinal variable by using the median as cut point (never (reference), <2 years, ≥2 years) for each digit of both codes. Tests for trend were performed by applying the Wald χ2 test to the duration variable as an ordinal variable. on October 23, 2024 by guest. Protected by copyright. http://oem.bmj.com/ m Given the broad age strata, we further adjusted for age at index date to avoid residual confounding by age (online supple- mental table S1). Adjusted analyses were limited to subjects with no missing data for the adjustment variables and complete occu- pational history (N=12 and 33 exclusions, respectively). Anal- yses were repeated for TGCT histological subtype. Associations heterogeneity was tested using polytomous logistic regression for matched case–control studies (SAS macro %subtype).27 P values for heterogeneity were derived from the likelihood ratio test.27 We additionally conducted sensitivity analyses: excluding cases with a personal history of cryptorchidism (N=40), and TGCT cases not confirmed by pathology reports (N=43). Statistical analysis Descriptive statistics (mean and SD for continuous and frequency and percentage for categorical variables) summarising partic- ipants’ characteristics were used. In the individual matching, some cases could not be matched to controls and vice versa. Therefore, we performed instead frequency matching on birth year grouped into 5-year categories and region of recruitment. Conditional logistic regression models were used to estimate the OR and 95% CI for ever being employed in a specific occupa- tion/industry compared with not (reference). Each of the jobs reported in the work history was considered. To obtain compa- rable exposure periods for cases and controls within 5-year age strata, we only considered exposures occurring before the index date (ie, date of TGCT diagnosis for cases, considering; the median age of cases at diagnosis in each stratum for controls). Data analysis used SAS statistical software V.9.4 (SAS Insti- tute), p values were two sided and considered statistically signif- icant if lower than 0.05. RESULTS h est. Protected by copyright. Covariates d h Protected by copyrig http://oem.bmj.com/ Occup Environ Med: first published as 10.1136/oemed-2022-108601 on 25 May 2023. Downloaded from Table 1 Characteristics of TGCT cases and controls (group A and group B), TESTIS study, N=1124 Guth M, et al. Occup Environ Med 2023;0:1–11. doi:10.1136/oemed-2022-108601 3 Workplace Characteristics TGCT cases (n=454) Group A controls (n=384) Group B controls (n=286) P value cases/all controls* P value controls A/controls B* n (%) n (%) n (%) Family history of TGCT <0.001 0.33 No 419 (92.3) 375 (97.7) 276 (96.5) Yes 33 (7.3) 7 (1.8) 9 (3.2) Missing 2 (0.4) 2 (0.5) 1 (0.4) Family history of cryptorchidism 0.01 0.76 No 422 (93) 369 (96.1) 276 (96.5) Yes 28 (6.2) 10 (2.6) 8 (2.8) Missing 4 (0.9) 5 (1.3) 2 (0.7) Age at voice change (years) 0.61 0.97 <12 16 (3.5) 17 (4.4) 9 (3.2) 12–16 375 (82.6) 301 (78.4) 231 (80.8) >16 53 (11.7) 56 (14.6) 38 (13.3) Missing 10 (2.2) 10 (2.6) 8 (2.8) Cannabis use at adolescence (12–17 years) 0.30 0.30 No 312 (68.7) 251 (65.4) 192 (67.1) Yes 142 (31.3) 133 (34.6) 94 (32.9) Cannabis use (18–25 years) 0.68 0.33 No 262 (57.71) 219 (57.03) 174 (60.84) Yes 192 (42.29) 164 (42.71) 112 (39.16) Missing 0 (0.0) 1 (0.26) 0 (0.0) Alcohol consumption 0.03 0.15 No 17 (1.52) 23 (3.43) 24 (3.58) Yes 429 (38.44) 361 (53.88) 262 (39.10) Boldface indicates p-value <0.05. P values from univariate conditional logistic regression models—except for age at diagnosis/inclusion, age at inclusion and year of birth which were matching factors. TGCT, testicular germ cell tumour. Table 1 continued Characteristics of the study population The mean age (±SD) of cases and group A and group B controls were 31.1 (±6.3), 33.3 (±5.6) and 32.9 (±5.2) years, respec- tively (table 1). Cases were younger than controls (p<0.001). No difference between the two control groups was observed, except for education level (p=0.002). Although most partici- pants were first born and single born, cases and controls differed for these characteristics (p=0.04 and 0.02, respectively), as well as for sibship size (p=0.03). Cases reported greater alcohol As the subjects included in the TESTIS study are young, the duration of employment is short. Thus, duration of employment Guth M, et al. Occup Environ Med 2023;0:1–11. Characteristics of the study population doi:10.1136/oemed-2022-108601 4 Workplace Table 2 ORs and 95% CIs for TGCT associated with occupations, overall and according to histological subtypes, TESTIS study Occupation description (ISCO-68) All TGCT cases Seminomas† Non-seminomas‡ P-HET§ Ca/Co (%ever) OR (95% CI)¶ Ca/Co (%ever) OR (95% CI) ¶ Ca/Co (%ever) OR (95% CI)¶ Professional, technical and related workers (0/1) 39.5/45.4 0.83 (0.63 to 1.08) 40.7/45.4 0.82 (0.59 to 1.15) 36.4/45.4 0.78 (0.53 to 1.14) 0.82 Architects, engineers and related technicians (0–2/0–3) 12.4/12.8 0.94 (0.63 to 1.40) 13.6/12.8 1.16 (0.72 to 1.89) 9.1/12.8 0.64 (0.34 to 1.21) 0.14 Doctors, dentists, veterinarians and similar workers (0–6/0–7) 4.0/6.9 0.58 (0.32 to 1.05) 3.3/6.9 0.48 (0.21 to 1.12) 6.1/6.9 0.83 (0.39 to 1.76) 0.34 Teachers (1–3) 6.9/8.8 0.83 (0.51 to 1.35) 6.5/8.8 0.65 (0.34 to 1.26) 6.7/8.8 0.89 (0.44 to 1.80) 0.53 Secondary education teachers (1–32) 4.5/4.3 1.09 (0.57 to 2.05) 4.7/4.3 0.89 (0.39 to 2.04) 4.2/4.3 1.29 (0.53 to 3.14) 0.56 Administrative and managerial workers (2) 12.9/16.3 0.86 (0.59 to 1.26) 15.0/16.3 0.97 (0.61 to 1.54) 9.7/16.3 0.75 (0.41 to 1.37) 0.51 Clerical and related workers (3) 20.5/19.5 1.02 (0.74 to 1.40) 21.5/19.5 0.99 (0.66 to 1.48) 18.8/19.5 0.98 (0.62 to 1.57) 0.99 Sales workers (4) 21.0/17.2 1.28 (0.92 to 1.77) 22.0/17.2 1.12 (0.74 to 1.67) 20/17.2 1.39 (0.87 to 2.20) 0.49 Salesmen, shop assistants and related workers (4–5) 13.1/8.2 1.66 (1.09 to 2.52) 12.6/8.2 1.29 (0.77 to 2.15) 12.7/8.2 1.91 (1.07 to 3.42) 0.32 Salesmen, shop assistants and demonstrators (4–51) 13.1/7.2 1.84 (1.20 to 2.82) 12.6/7.2 1.44 (0.86 to 2.43) 12.7/7.2 2.08 (1.16 to 3.75) 0.36 Other salesmen, shop assistants and demonstrators (4–51.90) 7.6/2.9 2.54 (1.38 to 4.65) 6.5/2.9 1.71 (0.82 to 3.57) 9.1/2.9 4.34 (2.00 to 9.42) 0.09 Service workers (5) 24.0/21.5 1.17 (0.86 to 1.58) 22.9/21.5 0.94 (0.64 to 1.39) 27.9/21.5 1.43 (0.94 to 2.16) 0.15 Cooks, waiters, bartenders and related workers (5–3) 10.2/8.9 1.21 (0.78 to 1.86) 9.3/8.9 0.95 (0.54 to 1.67) 13.3/8.9 1.50 (0.86 to 2.63) 0.26 Waiters, bartenders and related workers (5–32) 6.4/4.5 1.65 (0.94 to 2.90) 6.5/4.5 1.37 (0.69 to 2.74) 7.9/4.5 1.72 (0.83 to 3.57) 0.65 Waiter, general (5–32.10) 5.0/2.9 1.93 (0.99 to 3.74) 3.7/2.9 1.20 (0.49 to 2.90) 7.9/2.9 2.80 (1.29 to 6.09) 0.15 Protective service workers (5–8) 8.3/7.4 1.10 (0.69 to 1.77) 8.9/7.4 1.04 (0.58 to 1.87) 7.3/7.4 1.11 (0.55 to 2.24) 0.90 Firefighters (5–81) 3.1/1.7 1.59 (0.69 to 3.69) Agricultural, animal husbandry and forestry workers (6) 10.7/6.0 1.71 (1.07 to 2.72) 10.3/6.0 1.51 (0.85 to 2.68) 12.7/6.0 1.94 (1.05 to 3.61) 0.55 Farmers (6–1) 2.4/1.4 1.84 (0.71 to 4.76) Agricultural and animal husbandry workers (6–2) 8.6/4.8 1.71 (1.02 to 2.86) 6.5/4.8 1.25 (0.63 to 2.51) 12.1/4.8 2.12 (1.11 to 4.04) 0.27 Production and related workers, transport equipment operators and labourers (7/8/9) 48.6/40.8 1.35 (1.04 to 1.76) 46.3/40.8 1.23 (0.88 to 1.72) 51.5/40.8 1.43 (0.99 to 2.07) 0.56 Production supervisors and general foremen (7–0) 5.0/4.6 1.24 (0.69 to 2.25) 4.7/4.6 1.12 (0.52 to 2.43) 6.1/4.6 1.29 (0.59 to 2.83) 0.81 Machinery fitters, machine assemblers and precision-instrument makers (except electrical) (8–4) 13.8/8.8 1.72 (1.14 to 2.59) 13.6/8.8 1.74 (1.05 to 2.88) 13.3/8.8 1.41 (0.80 to 2.48) 0.59 Machinery fitters and machine assemblers (8–41) 3.6/1.7 2.46 (1.08 to 5.62) 2.3/1.7 1.81 (0.58 to 5.64) 3.0/1.7 1.58 (0.49 to 5.08) 0.87 Refrigeration and air-conditioning plan installer and mechanic (8–41.80) 2.6/1.2 2.43 (0.92 to 6.39) Motor-vehicle mechanics (8–43) 3.3/2.9 1.16 (0.56 to 2.39) Machinery fitters, machine assemblers and precision-instrument makers (except electrical) N.E.C. on October 23, 2024 by guest. Protected by copyright. http://oem.bmj.com/ n Med: first published as 10.1136/oemed-2022-108601 on 25 May 2023. Downloaded from supplemental table S1). The subcategory of ‘other salesmen, shop assistants and demonstrators’ (ISCO: 4–51.90) showed the strongest association, both with TGCT overall (OR 2.54; 95% CI (1.38 to 4.65)) and non-seminoma subtype (OR 4.34; 95% CI (2.00 to 9.42)), although CIs were wide. There was a positive association between TGCT (OR 1.71; 95% CI (1.02 to 2.82)), non-seminoma (OR 2.12; 95% CI (1.11 to 4.04)) and ‘agri- cultural and animal husbandry workers’ (ISCO: 6–2). A posi- tive association was further observed between TGCT risk and ‘production and related workers, transport equipment operators and labourers’ (ISCO: 7/8/9; OR 1.35; 95% CI (1.04 to 1.76)), whereof ‘machinery fitters, machine assemblers’ and ‘electrical consumption (p=0.03) and more testicular trauma (p=0.004) than controls. Family history of TGCT and of cryptorchidism were more frequent among cases than controls (p<0.001 and 0.01, respectively). Finally, there was no difference between cases and controls in age at voice change, smoking status, cannabis use, birth weight and gestational age. y guest. Protected by copyright. Characteristics of the study population (8–49) 7.6/4.9 1.60 (0.94 to 2.73) 7.5/4.9 1.58 (0.82 to 3.06) 8.5/4.9 1.64 (0.80 to 3.37) 0.94 Electrical fitters and related electrical and electronics workers (8–5) 10.0/5.7 1.80 (1.11 to 2.91) 9.3/5.7 1.65 (0.90 to 3.00) 10.3/5.7 1.71 (0.90 to 3.25) 0.94 Plumbers, welders, sheet-netal and structural metal preparers and erectors (8–7) 7.6/4.6 1.67 (0.97 to 2.87) 7.0/4.6 1.29 (0.65 to 2.55) 7.2/4.6 1.82 (0.86 to 3.83) 0.50 Welders and flame-cutters (8–72) 2.9/1.8 1.40 (0.59 to 3.29) 2.8/1.8 1.16 (0.40 to 3.36) 3.0/1.8 2.41 (0.74 to 7.81) 0.37 Sheet-metal workers (8–73) 1.4/1.2 1.31 (0.43 to 4.00) Painters (9–3) 1.9/2.2 0.70 (0.28 to 1.74) Bricklayers, carpenters and other construction workers (9-5) 9.5/7.5 1.27 (0.80 to 2.01) 8.4/7.5 1.19 (0.65 to 2.17) 10.9/7.5 1.38 (0.74 to 2.59) 0.74 Material handling and related equipment operators, dockers and freight handlers (9–7) 11.0/8.6 1.28 (0.83 to 1.97) 9.3/8.6 1.01 (0.57 to 1.79) 12.1/8.6 1.50 (0.83 to 2.69) 0.35 Transport equipment operators (9–8) 8.1/7.8 1.03 (0.64 to 1.65) 8.9/7.8 0.96 (0.53 to 1.73) 9.1/7.8 1.38 (0.73 to 2.62) 0.42 Laboureres N.E.C (9–9) 2.9/2.5 1.07 (0.48 to 2.38) Boldface indicates p-value <0.05. Total number of subjects presented for each code can vary due to the management of codes with missing digits. Subjects with a single job coded as missing data, were excluded from the analyses (N=33). †219 cases of seminoma TGCT were present in the TESTIS study. ‡191 cases of non-seminoma TGCT were present in the TESTIS study. §P value for heterogeneity derived from the likelihood ratio test, comparing seminoma versus non-seminoma tumours. ¶Estimates obtained comparing TGCT cases to group A and group B controls combined and adjusted for sibship size, being born from multiple pregnancy, personal history of testicular trauma, family history of TGCT and family history of cryptorchidism. Analysis was restricted to subjects with no missing data for the adjustment variables (N=12). Results presented if a job was held by more than five cases and five controls (grey line if less). Ca/Co (%ever), percentage of cases/controls ever employed; ISCO, International Standard Classification of Occupations; N.E.C, not elsewhere classified; P-HET, P-value for heterogeneity; TGCT, testicular germ cell tumour. TGCT risk by occupation (ISCO68) h b There was a positive association between TGCT risk and working as ‘salesmen, shop assistants and demonstrators’ (ISCO: 4–51; OR 1.84; 95% CI (1.20 to 2.82)), particularly for non-seminoma (ISCO: 4–51; OR 2.08; 95% CI (1.16 to 3.75)) (table 2, online Guth M, et al. Occup Environ Med 2023;0:1–11. doi:10.1136/oemed-2022-108601 Guth M, et al. Occup Environ Med 2023;0:1–11. TGCT risk by occupation (ISCO68) h b doi:10.1136/oemed-2022-108601 5 Table 3 ORs and 95% CIs for TGCT associated with occupations, according to employment duration (<2 vs ≥2 years), TESTIS study Occupation description (ISCO-68) Ca/Co (never) Ca/Co<2 Years OR1 (95% CI)† Ca/Co≥2 Years OR2 (95% CI)† P trend Professional, technical and related workers (0/1) 254/355 27/42 0.81 (0.48 to 1.39) 139/253 0.83 (0.62 to 1.10) 0.38 Architects, engineers and related technicians (0–2/0–3) 368/567 12/22 0.72 (0.33 to 1.50) 40/61 1.03 (0.66 to 1.62) 0.68 Teachers (1–3) 391/593 7/20 0.62 (0.25 to 1.50) 22/37 0.94 (0.53 to 1.68) 0.56 Administrative and managerial workers (2) 366/544 13/26 0.82 (0.41 to 1.68) 41/80 0.88 (0.57 to 1.34) 0.74 Clerical and related workers (3) 334/523 38/42 1.37 (0.85 to 2.22) 48/85 0.84 (0.87 to 1.26) 0.27 Sales workers (4) 332/538 30/37 1.28 (0.76 to 2.15) 58/75 1.28 (0.87 to 1.88) 0.34 Salesmen, shop assistants and related workers (4–5) 365/597 25/22 1.81 (0.98 to 3.33) 30/31 1.54 (0.90 to 2.66) 0.06 Salesmen, shop assistants and demonstrators (4–51) 365/603 25/18 2.12 (1.12 to 4.02) 30/29 1.65 (0.95 to 2.87) 0.02 Retail trade salesman (4–51.30) 391/622 16/13 1.77 (0.82 to 3.81) 13/15 1.33 (0.61 to 2.92) 0.27 Other salesmen, shop assistants and demonstrators (4–51.90) 388/631 22/9 3.58 (1.59 to 8.06) 10/10 1.55 (0.61 to 3.95) <0.01 Service workers (5) 319/510 32/37 1.31 (0.78 to 2.20) 69/103 1.11 (0.78 to 1.58) 0.53 Cooks, waiters, bartenders and related workers (5–3) 377/592 18/21 1.28 (0.66 to 2.51) 25/37 1.16 (0.67 to 2.00) 0.68 Waiters, bartenders and related workers (5–32) 393/621 13/9 2.58 (1.07 to 6.24) 14/20 1.22 (0.58 to 2.53) 0.10 Waiter, general (5–32.10) 399/631 13/7 3.38 (1.28 to 8.94) 8/12 1.11 (0.43 to 2.86) 0.05 Protective service workers (5–8) 385/602 7/10 1.11 (0.40 to 3.10) 28/38 1.10 (0.65 to 1.87) 0.92 Protective service workers N.E.C (5–89) 396/617 8/10 1.41 (0.52 to 3.79) 16/23 1.04 (0.52 to 2.04) 0.79 Watchman (5–89.40) 409/634 6/6 1.25 (0.37 to 4.22) 5/10 1.00 (0.34 to 2.99) 0.94 Agricultural, animal husbandry and forestry workers (6) 375/611 13/11 1.71 (0.73 to 3.98) 32/28 1.71 (0.99 to 2.95) 0.08 Agricultural and animal husbandry workers (6–2) 384/619 14/11 1.81 (0.78 to 4.19) 22/20 1.65 (0.86 to 3.15) 0.13 Production and related workers, transport equipment operators and labourers (7/8/9) 216/385 43/66 1.06 (0.68 to 1.66) 161/199 1.45 (1.09 to 1.94) 0.03 Production supervisors and general foremen (7–0) 399/620 6/13 0.78 (0.29 to 2.13) 15/17 1.62 (0.78 to 3.36) 0.38 Machinery fitters, machine assemblers and precision-instrument makers (except electrical) (8–4) 362/593 23/14 2.50 (1.24 to 5.06) 35/43 1.43 (0.87 to 2.34) 0.02 Motor-vehicle mechanics (8–43) 406/631 7/9 1.12 (0.39 to 3.17) 7/10 1.19 (0.44 to 3.26) 0.92 Machinery fitters, machine assemblers and precision-instrument makers (except electrical) N.E.C (8–49) 388/618 15/9 2.65 (1.12 to 6.26) 17/23 1.16 (0.59 to 2.30) 0.08 Electrical fitters and related electrical and electronics workers (8–5) 378/613 15/14 1.75 (0.81 to 3.76) 27/23 1.83 (1.01 to 3.32) 0.06 Electrical wiremen (8–55) 394/626 8/8 1.39 (0.50 to 3.88) 18/16 1.77 (0.87 to 3.60) 0.24 Plumbers, welders, sheet-metal and structural metal preparers and erectors (8–7) 388/620 16/10 2.23 (0.96 to 5.20) 16/20 1.36 (0.67 to 2.75) 0.13 Welders and flame-cutters (8–72) 408/638 7/6 1.31 (0.41 to 4.17) 5/6 1.51 (0.44 to 5.21) 0.73 Bricklayers, carpenters and other construction workers (9–5) 380/601 16/16 1.75 (0.84 to 3.67) 24/33 1.05 (0.60 to 1.86) 0.33 Material handling and related equipment operators, dockers and freight handlers (9–7) 374/594 26/31 1.39 (0.79 to 2.45) 20/25 1.15 (0.61 to 2.18) 0.49 Transport equipment operators (9–8) 386/599 16/29 0.81 (0.42 to 1.59) 18/22 1.31 (0.67 to 2.54) 0.60 Boldface indicates a p value <0.05. TGCT risk by occupation (ISCO68) h b Total number of subjects presented for each code can vary due to the management of codes with missing digits. Subjects with a single job coded as missing data, were excluded from the analyses (N=33). †Estimates obtained comparing TGCT cases to group A and group B controls combined and adjusted for sibship size, being born from multiple pregnancy, personal history of testicular trauma, family history of TGCT and family history of cryptorchidism. Analysis was restricted to subjects with no missing data for the adjustment variables (N=12). Results presented if a job was held by more than five cases and five controls. Ca/Co (ever)/(never), cases/controls ever/never employed; ISCO, International Standard Classification of Occupations; N.E.C, not elsewhere classified; TGCT, testicular germ cell tumour. http://oem.bmj.co Occup Environ Med: first published as 10.1136/oemed-2022-108601 on 25 May 2023. Downloaded from on October 23, 2024 by guest. Protected by copyright http://oem.bmj.com/ 23. Downloaded from p for trend=0.02) was observed. However, the CIs were wide, indicating substantial uncertainty around the risk estimates. fitters, related electrical, electronic workers’ (ISCO: 8–41; OR 2.46; 95% CI (1.08 to 5.62)/ISCO: 8–5; OR 1.80; 95% CI (1.11 to 2.91)). Finally, none of the observed associations showed heterogeneity between histologic subtypes. TGCT risk by industries (NAF99) Analyses by duration of employment (table 3) showed a posi- tive association between TGCT and subjects who had been employed for ≥2 years as ‘production and related workers, trans- port equipment operators and labourers’ (ISCO: 7/8/9; OR≥ 2 years=1.45; 95% CI (1.09 to 1.94); p for trend=0.03), whereof ‘electrical fitters and related electrical and electronics workers’, however, with a non-significant duration trend (ISCO: 8–5; OR≥2 years=1.83; 95% CI (1.01 to 3.32); p for trend=0.06). A positive association between TGCT risk and duration of employ- ment of less than 2 years for: ‘stock clerks’ (ISCO: 3–91; OR<2 years=2.27; 95% CI (1.14 to 4.50); p-for-trend=0.03); ‘other salesmen, shop assistants and demonstrators’ (ISCO: 4–51.90; OR< 2 years=3.58; CI (1.59 to 8.06); p for trend <0.01) and for ‘machinery fitters, machine assemblers and precision-instrument makers’ (ISCO: 8–4; OR<2 years=2.50; 95% CI (1.24 to 5.06); Analyses by duration of employment (table 3) showed a posi- tive association between TGCT and subjects who had been employed for ≥2 years as ‘production and related workers, trans- port equipment operators and labourers’ (ISCO: 7/8/9; OR≥ 2 years=1.45; 95% CI (1.09 to 1.94); p for trend=0.03), whereof ‘electrical fitters and related electrical and electronics workers’, however, with a non-significant duration trend (ISCO: 8–5; OR≥2 years=1.83; 95% CI (1.01 to 3.32); p for trend=0.06). A positive association between TGCT risk and duration of employ- ment of less than 2 years for: ‘stock clerks’ (ISCO: 3–91; OR<2 This analysis suggested an association between TGCT, non- seminoma and ‘agriculture, hunting and related service activities’ (NAF: 01) (table 4, online supplemental table S2). However, CIs were wide and compatible with inverse associ- ations. Although CIs were also wide, ORs suggested associa- tions between TGCT risk and ‘electrical installation work’, ‘installation of heating and air conditioning equipment’ (NAF: 45.3A; OR 1.98; 95% CI (1.12 to 3.50)/NAF: 45.3F; OR 2.95; 95% CI (1.18 to 7.39)). There was a positive associa- tion between TGCT and ‘trade, repair of motor vehicles and household goods’ (NAF: 50–52; OR 1.42; 95% CI (1.06 to 1.90)), ‘supermarkets’ (NAF: 52.1D; OR 2.28; 95% CI (1.28 to 4.06)), industries and ‘technical or professional secondary education’ (NAF: 80.2C; OR 1.55; 95% CI (1.03 to 2.34)). The analysis also suggested a positive association with ‘food guest. Protected by copyright. on October 23, 2024 by guest. Protected by copyright http://oem.bmj.com/ Occup Environ Med: first published as 10.1136/oemed-2022-108601 on 25 May 2023. Downloaded from Total number of subjects presented for each code can vary due to the management of codes with missing digits. Subjects with a single job coded as missing data, were excluded from the analyses (N=33). †Estimates obtained comparing TGCT cases to group A and group B controls combined and adjusted for sibship size, being born from multiple pregnancy, personal history of testicular trauma, family history of TGCT and family history of cryptorchidism. Analysis was restricted to subjects with no missing data for the adjustment variables (N=12). Results presented if a job was held by more than five cases and five controls (grey line if less). ‡219 cases of seminoma TGCT were present in the TESTIS study. §191 cases of non-seminoma TGCT were present in the TESTIS study. ¶ P-value for heterogeneity derived from the Likelihood Ratio Test, comparing seminoma versus non-seminoma tumours. Ca/Co (%ever), percentage of cases/controls ever employed; ISCO, International Standard Classification of Occupations; NAF-99, Nomenclature d’Activités Française-1999; N.E.C, not elsewhere classified; P-HET, P-value for heterogeneity; TGCT, testicular germ cell tumour. on October 23, 2024 by gue http://oem.bmj.com/ Occup Environ Med: first published as 10.1136/oemed-2022-108601 on 25 May 2023. Downloaded from p y ¶ P-value for heterogeneity derived from the Likelihood Ratio Test, comparing seminoma versus non-seminoma tumours. Ca/Co (%ever), percentage of cases/controls ever employed; ISCO, International Standard Classification of Occupations; NAF-99, Nomenclature d’Activités Française-1999; N.E.C, not elsewhere classified; P-HET, P-value for heterogeneity; TGCT, testicular germ cell tumour. the latter was found different from the association with non- seminoma (p for heterogeneity=0.04). retailing in specialty stores’ (NAF: 52.2; OR 2.89; 95% CI (1.03 to 8.14)) but the CI was wide, reflecting the statis- tical uncertainty of this risk estimate. The category of ‘other community, social and personal services’ (NAF: 90–93) was associated with non-seminoma (OR 2.26; 95% CI (1.32 to 3.87)). An inverse association was observed for non-seminoma for ‘other business activities’ (NAF: 74; OR 0.50; 95% CI (0.27 to 0.92)) with heterogeneity in this association (p for heterogeneity=0.04). ‘Hospital activities’ (NAF: 85.1A) were also inversely associated with TGCT (OR 0.56; 95% CI (0.32 to 0.98)) and seminoma (OR 0.33; 95% CI (0.13 to 0.81)); y guest. Protected by copyright. on October 23, 2024 by guest. Protected by copyright http://oem.bmj.com/ Occup Environ Med: first published as 10.1136/oemed-2022-108601 on 25 May 2023. Downloaded from Workplace retailing in specialty stores’ (NAF: 52.2; OR 2.89; 95% CI (1.03 to 8.14)) but the CI was wide, reflecting the statis- the latter was found different from the association with non- seminoma (p for heterogeneity=0.04). on October 23, 2024 by guest. Protected by copyright http://oem.bmj.com/ Occup Environ Med: first published as 10.1136/oemed-2022-108601 on 25 May 2023. Downloaded from Table 4 ORs and 95% CIs for TGCT associated with industries, overall and cording to histological subtypes, TESTIS study Industry description (NAF-99 code)* All TGCT cases Seminomas‡ Non-seminomas§ P-HET¶ Ca/Co (%ever) OR (95% CI)† Ca/Co (%ever) OR (95% CI)† Ca/Co (%ever) OR (95% CI)† Agriculture, hunting and forestry (01, 02) 7.9/5.2 1.43 (0.85 to 2.41) 7.5/5.3 1.32 (0.69 to 2.54) 9.8/5.3 1.64 (0.83 to 3.25) 0.65 Agriculture, hunting and related service activities (01) 7.6/4.8 1.56 (0.91 to 2.67) 7.5/4.8 1.48 (0.76 to 2.88) 9.1/4.8 1.79 (0.88 to 3.64) 0.70 Manufacturing (15–37) 31.3/29.4 1.10 (0.83 to 1.46) 31.3/29.4 1.13 (0.79 to 1.61) 29.3/29.4 1.00 (0.67 to 1.50) 0.66 Publishing, printing and reproduction of recorded media (22) 1.7/1.1 1.67 (0.56 to 4.99) Metalworking (28) 3.8/4.6 0.84 (0.44 to 1.61) 3.7/4.7 0.82 (0.35 to 1.89) 4.3/4.7 0.92 (0.37 to 2.34) 0.85 Electricity, gas and water supply (40, 41) 3.6/2.9 1.12 (0.53 to 2.36) 2.3/2.9 0.80 (0.28 to 2.28) 3.7/2.9 0.88 (0.32 to 2.40) 0.90 Electricity, gas, steam and hot water supply (40) 2.9/2.6 0.98 (0.44 to 2.19) 2.3/2.6 0.85 (0.29 to 2.46) 3.0/2.6 0.75 (0.25 to 2.25) 0.88 Construction (45) 20.5/16.6 1.27 (0.91 to 1.77) 16.8/16.4 0.94 (0.61 to 1.47) 22.6/16.4 1.41 (0.89 to 2.22) 0.22 Site preparation (45.1) 1.2/1.2 0.76 (0.23 to 2.50) Construction of building and civil engineering works (45.2) 6.2/4.7 1.38 (0.78 to 2.43) 3.3/4.7 0.61 (0.26 to 1.47) 9.1/4.7 1.80 (0.88 to 3.66) 0.06 Installation work (45.3) 11.2/6.4 1.99 (1.25 to 3.15) 9.4/6.4 1.52 (0.84 to 2.75) 9.8/6.4 1.59 (0.83 to 3.03) 0.92 Electrical installation work (45.3A) 6.9/4.04 1.98 (1.12 to 3.50) 6.6/4.1 1.66 (0.82 to 3.38) 6.1/4.1 1.96 (0.89 to 4.34) 0.76 Installation of heating and air conditioning equipment (45.3F) 3.3/1.2 2.95 (1.18 to 7.39) Finishing work (45.4) 5.0/5.3 0.87 (0.48 to 1.56) 4.7/5.1 0.82 (0.38 to 1.78) 6.1/5.1 1.15 (0.53 to 2.51) 0.54 Trade, repair of motor vehicles and household goods (50–52) 30.1/23.7 1.42 (1.06 to 1.90) 31.8/23.8 1.34 (0.94 to 1.92) 28.0/23.8 1.41 (0.93 to 2.14) 0.86 Motor trade and repair (50) 5.7/4.2 1.40 (0.78 to 2.52) 7.5/4.2 1.59 (0.81 to 3.12) 4.3/4.2 1.16 (0.47 to 2.86) 0.57 Wholesale trade and commercial intermediaries (51) 10.5/8.2 1.35 (0.87 to 2.10) 10.7/8.2 1.25 (0.73 to 2.16) 11.0/8.2 1.44 (0.79 to 2.65) 0.73 Retail trade and repair of household goods (52) 17.7/13.7 1.38 (0.97 to 1.96) 17.3/13.0 1.18 (0.76 to 1.82) 17.1/13.0 1.46 (0.90 to 2.39) 0.52 Retail sale in non-specialised shops (52.1) 7.4/4.3 1.91 (1.11 to 3.28) 5.6/4.3 1.29 (0.63 to 2.66) 9.8/4.3 3.00 (1.51 to 5.98) 0.10 Supermarkets (52.1D) 7.2/3.5 2.28 (1.28 to 4.06) 5.6/3.6 1.64 (0.78 to 3.46) 9.1/3.6 3.20 (1.55 to 6.60) 0.21 Food retail in specialised shops (52.2) 2.4/1.1 2.89 (1.03 to 8.14) Hotels and restaurants (55) 12.2/10.6 1.23 (0.82 to 1.84) 11.2/10.5 1.07 (0.63 to 1.79) 14.0/10.5 1.34 (0.79 to 2.29) 0.56 Transport and communications (60–64) 10.7/14.5 0.74 (0.50 to 1.09) 14.0/14.6 0.94 (0.59 to 1.50) 7.3/14.6 0.49 (0.26 to 0.94) 0.10 Financial activities (65–67) 4.8/5.4 1.05 (0.58 to 1.88) 4.7/5.4 0.95 (0.45 to 2.03) 6.1/5.4 1.72 (0.80 to 3.69) 0.29 Real estate, rental and business services (70–74) 24.1/30.2 0.77 (0.57 to 1.03) 29.0/30.3 0.91 (0.63 to 1.31) 17.1/30.3 0.51 (0.32 to 0.81) 0.05 Other business activities (74) 14.8/16.6 0.91 (0.64 to 1.30) 17.8/16.7 1.06 (0.69 to 1.63) 8.5/16.7 0.50 (0.27 to 0.92) 0.04 Public administration (75) 16.9/16.5 0.98 (0.69 to 1.38) 15.9/16.6 0.83 (0.52 to 1.30) 18.9/16.6 1.15 (0.71 to 1.86) 0.33 Education (80) 22.7/23.9 0.97 (0.72 to 1.31) 21.0/24.0 0.78 (0.52 to 1.15) 23.2/24.0 0.99 (0.65 to 1.51) 0.41 Secondary education (80.2) 15.8/12.3 1.33 (0.92 to 1.92) 15.0/12.4 1.03 (0.64 to 1.66) 15.2/12.4 1.27 (0.76 to 2.13) 0.55 General secondary education (80.2A) 3.1/3.3 0.88 (0.41 to 1.88) 2.8/3.3 0.50 (0.18 to 1.41) 3.0/3.3 1.33 (0.46 to 3.86) 0.20 Technical or professional secondary education (80.2C) 12.9/8.9 1.55 (1.03 to 2.34) 12.1/8.9 1.31 (0.78 to 2.21) 12.8/8.9 1.35 (0.77 to 2.39) 0.94 Health and social work (85) 7.4/12.9 0.57 (0.37 to 0.89) 8.4/13.0 0.64 (0.36 to 1.11) 7.9/13.0 0.61 (0.32 to 1.16) 0.92 Human health activities (85.1) 5.5/9.7 0.56 (0.33 to 0.93) 4.7/9.8 0.45 (0.22 to 0.93) 7.9/9.8 0.79 (0.41 to 1.52) 0.26 Hospital activities (85.1A) 4.5/7.9 0.56 (0.32 to 0.98) 2.8/7.9 0.33 (0.13 to 0.81) 7.9/7.9 1.01 (0.52 to 1.96) 0.04 Other community, social and personal services (90–93) 14.1/10.9 1.48 (1.00 to 2.19) 13.6/11.0 1.23 (0.75 to 2.01) 16.5/11.0 2.26 (1.32 to 3.87) 0.10 Recreational, cultural and sporting activities (92) 10.5/8.6 1.40 (0.91 to 2.17) 9.8/8.7 1.09 (0.62 to 1.90) 12.2/8.7 2.07 (1.14 to 3.76) 0.12 Boldface indicates a p value <0.05. TGCT risk by industries (NAF99) y ( <2 years=2.27; 95% CI (1.14 to 4.50); p-for-trend=0.03); ‘other salesmen, shop assistants and demonstrators’ (ISCO: 4–51.90; OR< 2 years=3.58; CI (1.59 to 8.06); p for trend <0.01) and for ‘machinery fitters, machine assemblers and precision-instrument makers’ (ISCO: 8–4; OR<2 years=2.50; 95% CI (1.24 to 5.06); Guth M, et al. Occup Environ Med 2023;0:1–11. doi:10.1136/oemed-2022-108601 6 Strengths and limitations This study has several strengths. Because of its national coverage (20 recruiting UH) and the participants’ detailed occupational history including tasks performed, it offers an opportunity to investigate and characterise occupations and industry sectors associated with testicular cancer in young adults in recent years. Detailed information allowed accounting for suspected or known TGCT risk factors. The observed increased TGCT risk for agricultural workers is consistent with previous studies.7 28–31 Conversely, two meta- analyses32 33 and a literature review34 of cancers among farmers found no evidence of an increased risk, which does not exclude the possibility that exposure to certain agricultural chemicals may be associated with increased TGCT risk.2 7 Several pesti- cides have been classified as EDCs due to their estrogenic or anti- androgenic effects.35 Exposure to EDCs has been hypothesised to increase TGCT risk by interfering with the regular hormonal balance.35 For instance, a positive association between dichlo- rodiphenyldichloroethylene, the most persistent metabolite of dichlorodiphenyltrichloroethane (DDT) and TGCT has been observed previously.2 Although DDT is now banned effectively in many countries, some older subjects may have been exposed early in their occupational history. While pesticides are the most commonly studied exposure in relation to TGCT among agricul- tural workers,7 12 they may also be exposed to a variety of other potentially carcinogenic substances, including solvents, metals, organic dusts and diesel exhaust fumes.36 This study has some limitations. on October 23, 2024 by guest. Protected by copyright http://oem.bmj.com/ Occup Environ Med: first published as 10.1136/oemed-2022-108601 on 25 May 2023. Downloaded from Analyses by duration of employment (online supplemental table S3) showed an association between TGCT risk and subjects who had been employed 2 years or more in ‘instal- lation work’ sector activities (NAF: 45.3; OR≥2 years=2.01; 95% CI (1.14 to 3.56); p for trend 0.02). Positive association between TGCT and employment less than 2 years was observed for ‘supermarket’ (NAF: 52.1D; OR< 2 years=2.97; 95% CI (1.33 to 6.63); p for trend=0.01). However, the CIs observed were also wide. Guth M, et al. Occup Environ Med 2023;0:1–11. doi:10.1136/oemed-2022-108601 7 Workplace on October 23, 2024 by guest. Protected by co http://oem.bmj.com/ Occup Environ Med: first published as 10.1136/oemed-2022-108601 on 25 May 2023. Downloaded from DISCUSSION Men ever employed in agricultural, production, transport and labourer occupations had an elevated TGCT risk compared with those who never worked there, and it appears to increase with duration of employment. An important strength of our study was to use two distinct classifications: one international (ISCO) for occupation and one French national (NAF) for industry that yielded consistent positive associations for several occupations/ industries, even if CIs were sometimes wider in one of the two. Thus, men employed in agricultural occupations showed an increased TGCT risk in both classifications, but with wider CI in NAF. Both classifications further found a positive association between electrical workers and TGCT. The positive association observed for salesmen was also supported by the association observed with the ‘retail in non-specialty stores’ and ‘super- market’ sectors for TGCT and non-seminoma risk. Finally, both classifications suggested an inverse association in relation to medical/paramedical workers, with wider CI regarding associa- tions found with ISCO. Finally, the results suggest that despite evolving regulation and increased workplace protection from carcinogen exposure,14 agricultural workers, production and transport workers such as electrical and electronic are consistently associated with TGCT over time. Nevertheless, humans are exposed to many chemicals, nearly 1500 are suspected EDCs, and this number will continue to grow.43 It is, therefore, necessary to conduct further epidemi- ological studies, with advanced methods of occupational expo- sure assessment, on the role of chemicals involved in high-risk occupations and TGCT risk, such as solvents or pesticides. Sensitivity analyses In contrast to our findings, an association between testicular cancer and firefighters has been suggested in several studies,39 40 potentially associated with some complex chemical compounds present in fire smoke.39 For example, firefighters may be more exposed to PFOA than the general population. A recent review meta-analysis suggests an average increase in TGCT risk per 10 ng/mL increase in serum PFOA.8 Other occupations have been found linked to TGCT risk in our study, although it may be more difficult to relate them to specific occupational exposures. Conversely to a previous study,31 our results suggest an inverse association of TGCT with health and social work. Furthermore, the observed excess TGCT risk in white-collar workers (ie, production supervisor and general foreman) is consistent with previous findings.7 41 However, this finding is possibly due to socioeconomic status (SES),31 yet the association between SES and TGCT risk remains unclear with divergent evidence.42 Most of the previously identified occupational groups/sectors were also identified when cases with a personal history of crypt- orchidism were excluded (N=40) (online supplemental table S4 and S5), when cases not confirmed by pathology reports were excluded (N=43) (online supplemental table S6 and S7) or when an adjustment for age at index date was made (online supplemental table S8 and S9). However, a numerically posi- tive association with ‘production and related workers, transport equipment operators and labourers’ (ISCO: 7/8/9) was observed when analyses were restricted to confirmed cases. Twitter Joachim Schüz @JoachimSchuz Twitter Joachim Schüz @JoachimSchuz Twitter Joachim Schüz @JoachimSchuz Acknowledgements We gratefully acknowledge the participants and their mothers for providing data, and medical practitioners for providing pathology reports. We are grateful to the medical teams of participating CECOS from the Fédération Française des CECOS, ART centres and maternity clinics that were involved in recruiting our participants and their mothers. We acknowledge the Direction de la Recherche Clinique from the Toulouse University hospital for their contribution to the coordination of the investigation centres. We thank the IPSOS Company for conducting telephone interviews. We acknowledge the contribution of Rémi Béranger to the TESTIS protocol and conduct of the TESTIS pilot study (TESTPERA). on October 23, 2024 by guest. Prote http://oem.bmj.com/ 1 on 25 May 2023. CONCLUSION l In conclusion, the findings suggest a possible role of some occu- pational exposures in the development of TGCT. Men employed in agricultural, electrical and electronics occupations had an elevated TGCT risk compared with those who never worked there, and it seems to be higher for workers employed for 2 years or more. The consistency of these findings with previous studies suggests opportunities to reinforce work health and safety measures to reduce occupational exposure to carcinogens. Also, further research targeting specific exposures is warranted to determine the role of agents or chemical currently involved in the identified high-risk occupations and investigate associated TGCT risk. Contributors AD, OP, HB, LB, JS, BC and BF participated in the conception and design of the study. AD, BDa, OP, HB, JS, LB, CM-G, SG’dE, MT, BDu, CF, BC and BF participated in the acquisition of data. MG, AC, ML, FD, AD, SA, AO, BC and BF participated in the analysis and interpretation of data. MG drafted the manuscript. AC, ML, AD, SA, AO, JS, BC and BF critically revised the manuscript for important intellectual content. All authors read and approved the final manuscript. BF is responsible for the overall content as a guarantor. Twitter Joachim Schüz @JoachimSchuz Downloaded from Collaborators Céline Chalas, Vanessa Gayet, Paul Pirtea, Pietro Santulli, Aurélie Vincent, Edouard Lecarpentier, François Goffinet, Dominique De Ziegler, Khaled Pocate, Virginie Barraud-Lange, Jean-Philippe Wolf, Emmanuel Dulioust, Nathalie Le Foll, Jacques Auger, Anne-Sophie Gille, Laurianne Kremer, Myriam Virlouvet, Lucile Ferreux, Guillemette Perier, Pauline Peretout, Diane Rivet, Véronique Drouineaud and Sandrine Rulle (Cochin Hospital, Paris); Rachel Levy, Nathalie Sermondade, Yassine Belaid, Marine Durand and Charlène Harbemont (Jean Verdier Hospital, Paris); Xavier Pollet-Villard, Vanina De Larouziere, Laurence Levy-Dutel, Florence Eustache and Isabelle Berthaut (Tenon Hospital, Paris); Jacqueline Saias-Magnan, Jeanne Perrin, Carole Daoud-Deveze and Laurent Nasca (La Conception Hospital, Marseille); Myriam Daudin, Nathalie Moinard, François Isus, Célia Bettiol and Laure Connan from Paule de Viguier Hospital in Toulouse; Laurent Janny, Valérie Bruhat, Florence Brugnon and Cyril Bouche (Estaing Hospital, Clermont-Ferrand); Isabelle Koscinski, Laëtitia Ladureau-Fritsch, Cécile Greze, Françoise Schmitt and Charles Pax (Obstetric medico-surgical centre, Strasbourg); Aline Papaxanthos, Clément Jimenez, Volcy Soula, Lucie Chansel and Olivier Delorme (Pellegrin maternity Hospital, Bordeaux); Pascale Dehee and Delphine Yalcinkaya (Femme-Mère-Enfant Hospital, Lyon); Céline Bouillon, Fabrice Guerif, Marie-Laure Couet, Véronique Ract, Olivia Gervereau, Elodie Poisson, Michel Lanoue, Anne Viallon and Catherine Guerin (Bretonneau Hospital, Tour); Julie Guitton, Marie Lefort and Valérie Mitchell (Calmette Hospital, Lille); Marie-Ange Clarotti, Ethel Szerman, Amélie Ancelle, Catherine Muris, Corinne Fourmy Chatel, Christine Denoual-Ziad, Claire De Vienne, Cécile Delesalle, Jean-Paul Bouiller and Antoine Clergeau (Caen Hospital, Caen); Oxana Blagosklonov, Alphée Bailly and Séverine Bey (Jean Minjoz Hospital, Besançon); Célia Ravel, Guilhem Jouve, Ségolène Veau, Laurent Vandenbroucke and Agnès Letremy (South Hospital, Rennes); Patricia Fauque and Julie Barberet (Dijon Hospital, Dijon); Stéphanie Lattes, Emmanuelle Thibault, Fabienne Bernardin, Pierre Besnier and Clémence Martin (l’Archet Hospital, Nice); Catherine Diligent, Françoise Touati, Nicolas Monnin and Christel Hersant (regional University maternity Hospital, Nancy); Vanessa Loup-Cabaniols, Alice Ferrieres, Anna Gala, Elodie Scalici, Lucile Sablayrolles, Tiffany Mullet, Audrey Chabert, Christelle Saintpeyre, Mélanie Caro, Michèle Nou and Marie Sicard (Arnaud de Villeneuve Hospital, Montpellier); Sylvianne Hennebicq, Pascale Hoffmann, Claire Thomas-Cadi, Nicole Quenard, Evelyne Warembourg, Laure Villaret and Julien Bessonnat (Couple-Enfant Hospital, Grenoble); Marie-Claude Blocquaux, Frédérique Carre-Pigeon, Béatrice Delepine, Olivier Graesslin and Julie Burette (Maison Blanche Hospital, Reims); Rémi Béranger and Aude Fléchon. on October 23, 2024 by guest. Protected by copyright. http://oem.bmj.com/ m Strengths and limitations doi:10.1136/oemed-2022-108601 8 Workplace on October 23, 2024 by guest. Protected by co http://oem.bmj.com/ Occup Environ Med: first published as 10.1136/oemed-2022-108601 on 25 May 2023. Downloaded from observed were on jobs for which we had plausible hypotheses, and that have been found in previous studies. There is also a risk of bias due to differential participation in case–control studies. This bias is positively associated with the difference in response rates between groups for the characteristic causing differential participation, and inversely associated with the total response rate in controls in previous studies.45 However, status of non- participants (N=96 people who were contacted but did not participate in the study, see online supplemental figure S1) was unfortunately not available to compare response rates between cases and controls. The overall response rate in this study is like that of previous studies.46–48 Yet, in the clinical setting, it is likely that additional eligible cases and controls were not interested in participating but not reported by the centres, and our response rate may be overestimated. A previous French hospital-based case–control study on TGCT risk factors, with recruitment of cases in CECOS at time of sperm cryopreservation (between 2002 and 2005), reported a response rate of 81% for cases and of 39% for controls recruited among partners of women in the maternity clinic of the same hospital as the CECOS.49 Our occu- pational data were self-reported and could have been subject to recall bias. However, the use of structured documents to prepare the interview and questionnaires, as well as training of inter- viewers and telephone interviews blinded to case–control status were intended to minimise recall bias. Thus, it is more likely that recall bias was non-differential with respect to recall of life- time work history and coding by an industrial hygienist blinded to case–control status, leading instead to null bias.50 There is also a difference in the regional distribution of the population, with participating controls more often coming from nearby/ urban locations. Some types of jobs may be underrepresented for our control population, such as agricultural jobs, leading to a potential differential job classification error and thus biasing the OR.50 Results by duration of employment should also be inter- preted with caution. The subjects’ young age resulted in a low median duration of employment, which may have reduced the strength of the association in this analysis. Strengths and limitations The subtypes show different peak incidences by age (ie, the highest incidence rate of non-seminoma is in the 25–29 years age group, while it is in the 35–39 years age group for seminomas2), so the results may be biased by analysing the duration for all TGCT, when we observed a stronger effect in one of the subtypes. We could not verify the influence of histological subtype on risk by duration due to an insufficient number of subjects. 4Environment and Lifestyle Epidemiology Branch, International Agency for Research on Cancer/World Health Organization (IARC/WHO), Lyon, France 5Department of Medical Oncology, Centre Léon Bérard, Lyon, France 6DEFE (Développement Embryonnaire, Fertilité, Environnement) INSERM 1202 Universités Montpellier et Toulouse 3, CECOS Hôpital Paule de Viguier, CHU de Toulouse, Toulouse, France observed were on jobs for which we had plausible hypotheses, and that have been found in previous studies. There is also a risk of bias due to differential participation in case–control studies. This bias is positively associated with the difference in response rates between groups for the characteristic causing differential participation, and inversely associated with the total response rate in controls in previous studies.45 However, status of non- participants (N=96 people who were contacted but did not participate in the study, see online supplemental figure S1) was unfortunately not available to compare response rates between cases and controls. The overall response rate in this study is like that of previous studies.46–48 Yet, in the clinical setting, it is likely that additional eligible cases and controls were not interested in participating but not reported by the centres, and our response rate may be overestimated. A previous French hospital-based case–control study on TGCT risk factors, with recruitment of cases in CECOS at time of sperm cryopreservation (between 2002 and 2005), reported a response rate of 81% for cases and of 39% for controls recruited among partners of women in the maternity clinic of the same hospital as the CECOS.49 Our occu- pational data were self-reported and could have been subject to recall bias. However, the use of structured documents to prepare the interview and questionnaires, as well as training of inter- viewers and telephone interviews blinded to case–control status were intended to minimise recall bias. Strengths and limitations Thus, it is more likely that recall bias was non-differential with respect to recall of life- time work history and coding by an industrial hygienist blinded to case–control status, leading instead to null bias.50 There is also a difference in the regional distribution of the population, with participating controls more often coming from nearby/ urban locations. Some types of jobs may be underrepresented for our control population, such as agricultural jobs, leading to a potential differential job classification error and thus biasing the OR.50 Results by duration of employment should also be inter- preted with caution. The subjects’ young age resulted in a low median duration of employment, which may have reduced the strength of the association in this analysis. The subtypes show different peak incidences by age (ie, the highest incidence rate of non-seminoma is in the 25–29 years age group, while it is in the 35–39 years age group for seminomas2), so the results may be biased by analysing the duration for all TGCT, when we observed a stronger effect in one of the subtypes. We could not verify the influence of histological subtype on risk by duration due to an insufficient number of subjects. 7Fédération Française des CECOS, Paris, France 8 8Centre Clinico-Biologique d’AMP-CECOS, AP-HM La Conception University Hospital, Marseille, France 8Centre Clinico-Biologique d’AMP-CECOS, AP-HM La Conception University Hospital, Marseille, France 9 9CECOS de Lyon, Service de Médecine de la Reproduction, Hôpital Femme Mère Enfant, Bron, France 9CECOS de Lyon, Service de Médecine de la Reproduction, Hôpital Femme Mère Enfant, Bron, France 10Department of Functional Genomics and Cancer, Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch-Grafenstaden, France 11 10Department of Functional Genomics and Cancer, Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch-Grafenstaden, France 11 10Department of Functional Genomics and Cancer, Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch-Grafenstaden, France 11 Biologie Moléculaire et Cellulaire, Illkirch-Grafenstaden, France 11 11CECOS Nord Lille, Hôpital Albert Calmette, Lille, France 12 11CECOS Nord Lille, Hôpital Albert Calmette, Lille, France 12 d l d l d 12Médecine et Biologie de la Reproduction-CECOS, CHU Bretonneau, Tours, France 13 12Médecine et Biologie de la Reproduction-CECOS, CHU Bretonneau, Tours, France 13 13Service des Maladies Professionnelles, Hospices Civils de Lyon, Pierre Bénite, France 13Service des Maladies Professionnelles, Hospices Civils de Lyon, Pierre Bénite, France 13Service des Maladies Professionnelles, Hospices Civils de Lyon, Pierre Bénite, F Guth M, et al. Occup Environ Med 2023;0:1–11. doi:10.1136/oemed-2022-108601 Author affiliations 1UMRESTTE, Université Claude Bernard Lyon 1, Lyon, France 2Radiation: Defense, Health, Environment, INSERM UMR1296, Lyon, France 3Prevention Cancer Environnement Departement, Centre Léon Bérard, Lyon, France Strengths and limitations The main one was the diffi- culty to obtain an unbiased random sample of general population controls given the low response rate in the target population.19 20 Another limitation was our inability to determine the controls’ representativeness of the source population from which the cases originated; this limitation has been discussed in detail previ- ously.17 While it has been suggested that occupational exposure might not be relevant for TGCT because of the young age of patients and the short duration of occupational exposure, the presence of GCNIS early in life and TCGT in young adulthood supports the hypothesis of combined early and later life expo- sures in the development of TGCT.2 Although this limitation is not specific to our study, the method applied identified occu- pational groups with higher risk due to collective exposure to agents or chemicals, exposure to these also varies within each job or industry, which is not accounted for in this way.12 Grouping potentially highly exposed subjects with unexposed from the same job could attenuate the strength of the association. Based on a priori hypothesis, we investigated associations between TGCT and occupations/industries. The use of two complemen- tary nomenclatures, based on broad occupation/industry cate- gories and associated subcategories, involved multiple analyses. Yet, as these variables are related, as well as to allow interpre- tation of results in light of previous literature, correction for multiple testing and the commonly used Bonferroni method were not considered appropriate.44 Therefore, positive asso- ciations may have occurred by chance and our results should be interpreted with caution. Of note, most of the associations g An increased TGCT risk in non-agricultural workers such as production and related workers, transportation equipment oper- ators, and labourers has also been observed, especially for those who have held these jobs for 2 years or more. 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Open access This is an open access article distributed under the terms of the Creative Commons Attribution IGO License (CC BY 3.0 IGO), which permits use, distribution,and reproduction in any medium, provided the original work is properly cited. In any reproduction of this article there should not be any suggestion that WHO or this article endorse any specific organization or products. The use of the WHO logo is not permitted. This notice should be preserved along with the article’s original URL 26 Hodges-Simeon CR, Gurven M, Cárdenas RA, et al. Voice change as a new measure of male pubertal timing: a study among Bolivian adolescents. Ann Hum Biol 2013;40:209–19. 27 Wang M, Spiegelman D, Kuchiba A, et al. Statistical methods for studying disease subtype heterogeneity. Stat Med 2016;35:782–800. 28 Hardell L, Näsman A, Ohlson CG. Case-control study on risk factors for Testicular cancer. Int J Oncol 1988. on October 23, 2024 by guest. 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https://openalex.org/W2763745409	https://europepmc.org/articles/pmc5635499?pdf=render	English	null	Mortality in patients treated with intravitreal bevacizumab for age-related macular degeneration	BMC ophthalmology	2,017	cc-by	4,516	© The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Abstract Background: The aim of this study is to analyze mortality in patients treated with bevacizumab for wet AMD. Methods: We conducted a retrospective case-control study between patients who received intravitreal injections of bevacizumab as the sole treatment for exudative AMD between September 2008 and October 2014 (n = 5385) and age and gender matched controls (n = 10,756). All individuals included in the study were reviewed for sociodemographic data and comorbidities. Survival analysis was performed using adjusted Cox regression, using relevant adjusted variables. Results: During follow-up (maximum: 73 months), 1063 (19.7%) individuals after bevacizumab died compared with 1298 (12.1%) in the control group (P < .001). After adjusted Cox survival regression, mortality differed significantly between the groups, Odds ratio = 1.69, (95% C.I. 1.54–1.84), P < .001. Conclusions: We found an increased long-term mortality in individuals with wet AMD treated with bevacizumab compared to a same age and gender group without wet AMD. Keywords: Neovascular AMD, Anti-VEGF, Bevacizumab, Safety, Mortality Keywords: Neovascular AMD, Anti-VEGF, Bevacizumab, Safety, Mortality Mortality in patients treated with intravitreal bevacizumab for age-related macular degeneration Joel Hanhart1, Doron S. Comaneshter2, Yossi Freier Dror3 and Shlomo Vinker2,4 Correspondence: hanhart@szmc.org.il 1Department of Ophthalmology, Shaare Zedek Medical Center, 12 Beyt Street, 91031 Jerusalem, Israel Full list of author information is available at the end of the article Hanhart et al. BMC Ophthalmology (2017) 17:189 DOI 10.1186/s12886-017-0586-0 Hanhart et al. BMC Ophthalmology (2017) 17:189 DOI 10.1186/s12886-017-0586-0 Background VEGF serum concentration, to the extent that 1 month after the treatment, VEGF serum level is only 23% of baseline [3]. Circulating VEGF protects vascular patency and integrity [4]. Intravitreal anti-vascular endothelial growth factor (anti- VEGF) treatment has revolutionized the management of many retinal conditions, including age-related macular degeneration (AMD). Several anti-VEGF agents are used in the treatment of neovascular AMD. Ranibizumab and aflibercept are approved as ophthalmic therapies. Bevaci- zumab is a full-length humanized monoclonal IgG anti- body of 149 kDa that inhibits all VEGF-A isoforms [1]. Approved in 2004 by the FDA, for systemic use in the treatment of certain metastatic cancers, bevacizumab is widely used off-label as intravitreal therapy in neovascu- lar AMD since its efficacy was described more than a decade ago [2]. In prospective studies of bevacizumab treatment for neo- vascular AMD, mortality was found to be 0.81%–10.00% at 1 year [5–10] and 5.07%–5.97% at 2 years [11, 12]. A recent meta-analysis of 6 randomized controlled trials reported that approximately 25% more bevacizumab-treated than ranibizumab-treated patients experienced one or more ser- ious non-ocular adverse events over one and 2 years. Among patients who received bevacizumab, overall mortal- ity was 1.95% at 1 year (25/1282 patients) and 5.78% at 2 years (51/882) [13]. Another meta-analysis that comprised 1623 patients reported 1.91% mortality at 1 year [14]. How- ever, many published studies and meta-analyses were not powered enough to accurately assess the systemic risks of anti-VEGF intravitreal injections [15]. Reduced systemic VEGF level was demonstrated in pa- tients who received intravitreal anti-VEGF agents, the systemic effect was most obvious with bevacizumab. In- traocular injection of bevacizumab strongly decreases In the public health system in Israel, patients diag- nosed with neovascular AMD are offered bevacizumab as a first line agent, in accordance with the efficacy dem- onstrated by major studies [5, 7–9, 11]. * Correspondence: hanhart@szmc.org.il 1Department of Ophthalmology, Shaare Zedek Medical Center, 12 Beyt Street, 91031 Jerusalem, Israel Full list of author information is available at the end of the article Page 2 of 6 Hanhart et al. BMC Ophthalmology (2017) 17:189 Page 2 of 6 Page 2 of 6 We report the mortality of all patients treated during a 6 year period, with intravitreal bevacizumab for neovascu- lar AMD, in the largest health maintenance organization in Israel; and compare it to the mortality of age and gender-matched individuals not-exposed to bevacizumab. Background 29 since high myopia could not be ruled out as the cause of choroidal neovascularisation; 18 as the reason for treatment may have been a concomitant diagnosis of ret- inal vein occlusion; in 4 patients, inflammatory condi- tions were identified as the possible etiology of choroidal neovascularisation. Patients who received other intraoc- ular anti-VEGF agents (pegaptanib, ranibizumab, afliber- cept) or systemic anti-VEGF therapy at any time were excluded from the analysis. Statistical analysis For all ratio variables, means and standard deviations were calculated and baseline differences between the groups evaluated using a t-test. For all nominal variables, absolute frequencies and percentages were calculated and baseline differences between the groups were assessed using a Chi-square test. The socioeconomic or- dinal variable baseline differences between the groups were evaluated using the Mann-Whitney test. To com- pare mortality over time between the groups, survival ana- lysis was performed using adjusted Cox regression. The dependent variable was survival. The time-dependent co- variate for the treatment group (bevacizumab) was the interval between the first injection to survival or death; and for the control group, the interval between the start of monitoring (date of first injection in the corresponding bevacizumab treated patient) to survival or death, all trun- cated at 7 years. Adjusted variables were age, smoking, al- cohol abuse, hypertension, diabetes, obesity, congested heart failure, liver cancer, ischemic heart disease, and cere- brovascular accident. For all individuals included in the analysis, we ex- tracted information from the registry regarding the fol- lowing conditions, which have been reported to be more prevalent in AMD patients and to be associated with increased mortality [17–19]: smoking, alcohol abuse, is- chemic heart disease, cerebrovascular disease, congestive heart failure, liver cancer, obesity, and (unilateral/bilat- eral) pseudophakia. The definitions in the Clalit database of alcohol abuse are based on the Diagnostic and Statistical Manual of Mental Disorders, version IV. Cerebrovascular disease was diagnosed following the criteria of the National In- stitute of Neurological Disorders [20]. The clinical data standards of the American College of Cardiology/Ameri- can Heart Association Task Force were used to define congestive heart failure and ischemic heart disease [21]. A body mass index of 30 kg/m2 or higher defined obesity. Additional information extracted from patients’ files included age, gender, marital status, and socioeconomic status. Statistical analyses were conducted using the SPSS statistical software (Version 20). The criterion for accepting the research hypothesis was: Alpha (α) = .05 (one-sided). The criterion for negating the preliminary differences between the treatment and the control group was: Alpha (α) = .05 (two-sided). The date of death was automatically communicated from the Israeli Interior Ministry via the unique national identity number. The cause of death was not recorded. Ethics approval was obtained from the Ethics Commit- tee of the Clalit Health Services. Data sources This retrospective, population-based analysis accessed data from the electronic medical records of all individ- uals affiliated with Clalit Health Services who received intravitreal injections of bevacizumab for treatment of AMD between September 2008 and October 2014. For each wet AMD patient treated with bevacizumab in the study group, two individuals were matched in age and gender from the members of Clalit Health Services. A matched control had the same age as the case on the date of first bevacizumab injection. Criteria for this reference group were no recorded exposure to anti-VEGF and con- tinual membership in Clalit Health Services from Septem- ber 2008 until October 2014, excepting death. Clalit Health Services maintains a chronic disease registry database that includes information collected from a variety of sources: primary care physician reports, medication-use files, hospitalization records, and out- patient clinic records. The methods of registry acquisi- tion and maintenance were described by Rennert and Peterburg [16]. Study population A total of 5385 individuals met the criteria established for the treatment group; and 10,756 aged and gender matched individuals comprised (the control group). In the nationwide Clalit Health Services records, we identified patients treated by anti-VEGF for wet AMD, and excluded those for whom there was doubt regarding the indication of the treatment. Forty-seven patients were excluded because it was not possible to eliminate diabetic macular oedema as the indication for injections; Sociodemographic and clinical characteristics of the groups are shown in Table 1. Patients in the treatment group were a mean 3.5 months older than controls (81.2 vs. 80.9 years). The proportion of males was the same, Hanhart et al. BMC Ophthalmology (2017) 17:189 Page 3 of 6 Table 1 Patient characteristics and outcome Table 1 Patient characteristics and outcome Treated with bevacizumab (N = 5385) N (%) Not treated with bevacizumab (N = 10,756) N (%) P-value Age Start [mean ± SD] 81.17 ± 8.91 80.88 ± 8.91 .051 Male 2460 (45.7) 4916 (45.7) .979 Married 2180 (40.5) 4637 (43.1) < .001 Socioeconomic statusa <.001 High 1213 (22.6) 2910 (27.1) Medium 2510 (46.7) 4620 (43.1) Low 1652 (30.7) 3190 (29.8) Cataract 2353 (43.7) 3585 (35.9) < .001 Smoking 1008 (18.7) 1555 (14.5) < .001 Alcohol 27 (.05) 64 (.06) .524 Hypertension 4142 (76.9) 7666 (71.3) < .001 Diabetes mellitus 1821 (33.8) 3019 (28.1) < .001 Obesity 1413 (26.2) 2581 (24.0) < .001 Congestive heart failure 538 (10.0) 893 (8.3) < .001 Liver cancer 4 (.01) 6 (.01) .913 Ischemic heart disease 2030 (37.7) 3434 (31.9) < .001 Cerebrovascular accident 857 (15.9) 1508 (14.0) < .001 Mortality 1063 (19.7) 1298 (12.1) < .001 aMann-Whitney test long-term adverse effects observed for bevacizumab. However, this is difficult to detect in patients who re- ceive intravenous doses of bevacizumab, due to the re- duced survival inherent to the malignant condition being treated; nevertheless, apprehensions have been raised [23]. 45.7% in both groups. A high prevalence of medical co- morbidities was found in both groups, though higher in the bevacizumab group. During follow-up (maximal follow-up of 73 months), 1063 (19.7%) patients who used bevacizumab died, com- pared to 1298 (12.1%) in the control group (P < .001). Cumulative survival was greater in the control group (Table 2, Fig. 1). After adjusted Cox regression, mortality was greater for the treatment group, OR = 1.69, (95% C.I. 1.54–1.84), P < .001(Table 3). Study population In the current study, risk factors previously reported to be common to AMD and cardiovascular disease [24– 26] were found to account in part for the increased mor- tality of the bevacizumab users. Nevertheless, the in- creased mortality persisted after adjusting for cardiovascular risk factors. Mortality specifically associ- ated with wet AMD has been attributed to the visual im- pairment it induces [27]. Anti-VEGF treatments have been demonstrated to restore vision [2, 5, 7–13, 28, 29]. Hence, our results support the contribution of bevacizu- mab to increased mortality, beyond the condition of wet AMD. The mean number of injections was significantly lower in patients who died 6.1 (6.43) vs. survived 8.3 (8.82) years, t(2158) = 8.98, P < .001. Patients died after having being treated during 10.50 ± 13.57 months, 18.8 ± 16.80 months after the last injection. Discussion We report increased mortality in patients treated with bevacizumab for wet AMD, compared to age and gender matched individuals for whom there was no record of a prescription to any anti-VEGF agent. It must be noted that, in our study, cases and controls differ on just about every risk factor for death (Table 1). If inclusion of categorical and quantitative variables did not fully capture the association between the factors and death, then there may be residual confounding. A con- trol group that would circumvent those methodologic is- sues would consist of patients with wet AMD who did not receive injections. In our era, this population does not exist. Comparing two populations with wet AMD in different periods would introduce other serious bias. Bevacizumab is known to escape the eye, reach the general circulation, and inhibit systemic VEGF-A [3]. VEGF-A is involved in homeostasis and healing in many systems. As an anti-angiogenic agent, bevacizumab may impair the ability of vascular tissues to contribute to healing [4, 22]. Impairment of normal healing rather than direct injury to vital organs seems to explain the Hanhart et al. BMC Ophthalmology (2017) 17:189 Page 4 of 6 Table 2 Survival (Life Table) Beva- cizumab Year Entering Interval Withdrawing during Interval Exposed to Risk Terminal Events Proportion Terminating Cumulative Proportion Surviving at End of Interval No 0–1 10,756 3050 9231 517 .06 .94 1–2 7189 2336 6021 327 .05 .89 2–3 4526 1443 3805 219 .06 .84 3–4 2864 963 2383 124 .05 .80 4–5 1777 770 1392 73 .05 .76 5–6 934 611 629 32 .05 .72 6–7 291 285 149 6 .04 .69 Yes 0–1 5385 1527 4622 470 .10 .90 1–2 3388 1099 2839 238 .08 .82 2–3 2051 631 1736 168 .10 .74 3–4 1252 407 1049 99 .09 .67 4–5 746 313 590 63 .11 .60 5–6 370 249 246 21 .09 .55 6–7 100 96 52 4 .08 .51 There would be two major flaws if we wanted to compare AMD patients without neovascularization with patients having the neovascular form. First, information extracted from such electronic medical records lack the precision required to be certain that patients registred as having dry AMD do not suffer from the neovascular form in at least one of their eyes. Discussion Then, since wet and dry AMD do not necessarily share the same risk profile [26, 30, 31], the risk of confounding by indication would persist. A limitation of this study is that our database does not differentiate between unilateral and bilateral injections, and provides only limited information on ocular condi- tions. Visual acuity, for instance, is not recorded. Pa- tients who went on to use other anti-VEGF treatments were excluded from this study. This might introduce a bias into the comparison death rates, as indivuals receiv- ing second line treatments do not necessarily share the same risk profile as people responding to bevacizumab. Fig. 1 Cumulative Survival. Cumulative survival is greater in patients who were not exposed to bevacizumab Fig. 1 Cumulative Survival. Cumulative survival is greater in patients who were not exposed to bevacizumab Hanhart et al. BMC Ophthalmology (2017) 17:189 Page 5 of 6 Page 5 of 6 Funding None. Availability of data and materials The data that support the findings of this study are available from Kupat Cholim Clalit but restrictions apply to the availability of these data, which were used under license for the current study, and so are not publicly available. Data are however available from the authors upon reasonable request and with permission of Kupat Cholim Clalit. Authors’ contributions DSC was involved in the acquisition and analysis of the data, YFD in statistical analysis of the data, SV in designing the study and revising the manuscript. JH designed the study, overviewed statistical analysis and drafted the manuscript. All authors made substantial contributions to this study and gave final approval for its submission. All authors read and approved final manuscript. Ethics approval and consent to participate Ethics approval was obtained from the Ethics Committee of the Clalit Healt Services. Consent for publication Not applicable. Table 3 Mortality (Cox regression) Variables B S.E. Competing interests No relevant disclosures for for Doron S Comaneshter, Yossi Freier Dror and Shlomo Vinker. Joel Hanhart has given lectures on diabetic macular edema for Novartis. Another weakness of our data is that the cause of death is not available. Nevertheless, all-cause mortality has some advantages as a principle end point, given the potential for misclassifying the cause of death [32]. Conclusions The findings presented raise questions regarding the use of bevacizumab for wet AMD. Other anti-VEGF intraoc- ular compounds are used as second-line therapy in Israel. Due to the observed delay between the last beva- cizumab injection and death, our data do not enable valid assessment of the effects of ranibizumab and afli- bercept on mortality. Additional data is needed to cor- roborate our worrying observation that bevacizumab intraocular injections may be associated with increased mortality. If confounding by indication could be ruled out but economic reasons precluded immediate inter- ruption of bevacizumab therapy for wet AMD, it would be crucial to define groups of higher and lower risk, to enable physicians and patients to discuss the systemic impact of ocular therapy and adequately balance ex- pected gains and risks. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 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AMD: Age-related macular degeneration; FDA: Food and drug administration; VEGF: vascular endothelial growth factor Discussion Wald (df = 1) p-value OR Gender (male) .053 .043 1.507 .220 1.054 Age start (Year) .013 .004 12.927 .000 1.013 Socioeconomic (low) .083 .057 2.093 .148 1.086 Smoking .162 .061 7.135 .008 1.176 Alcohol .323 .240 1.809 .179 1.381 Hypertension .032 .056 .322 .570 1.032 Diabetes .015 .046 .106 .745 1.015 Obesity .071 .054 1.724 .189 1.073 Congestive heart failure .172 .055 9.779 .002 1.187 Liver cancer .775 .516 2.255 .133 2.170 Ischemic heart disease .000 .046 .000 .993 1.000 Cerebrovascular accident .153 .051 9.065 .003 1.166 Bevacizumab use .527 .043 153.744 .000 1.694 Availability of data and materials However, to our knowledge, such a difference has never been reported. Acknowledgements Cindy Cohen provided editing services. 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(Chicago, Ill : 1960). 2004;122(5):716–26. 26. Clemons TE, Kurinij N, Sperduto RD. Associations of mortality with ocular disorders and an intervention of high-dose antioxidants and zinc in the age-related eye disease study: AREDS report no. 13. Arch. Ophthalmol. (Chicago, Ill : 1960). 2004;122(5):716–26.
https://openalex.org/W2913686851	https://boris.unibe.ch/127029/1/New%20Viruses%20from%20the%20Ectoparasite%20Mite%20Varroa%20destructor%20Infesting%20Apis%20mellifera%20and%20Apis%20cerana.pdf	English	null	New Viruses from the Ectoparasite Mite Varroa destructor Infesting Apis mellifera and Apis cerana	Viruses	2,019	cc-by	11,721	Received: 1 November 2018; Accepted: 22 January 2019; Published: 24 January 2019 Abstract: Varroa destructor is an ectoparasitic mite of Asian or Eastern honeybees Apis cerana (A. cerana) which has become a serious threat to European subspecies of Western honeybees Apis mellifera (A. mellifera) within the last century. V. destructor and its vectored honeybee viruses became serious threats for colony survival. This is a short period for pathogen- and host-populations to adapt. To look for possible variation in the composition of viral populations we performed RNA metagenomic analysis of the Western honeybee subspecies A. m. ligustica, A. m. syriaca, A. m. intermissa, and A. cerana and their respective V. destructor mites. The analysis revealed two novel viruses: Varroa orthomyxovirus-1 (VOV-1) in A. mellifera and V. destructor and a Hubei like-virga virus-14 homolog in V. destructor. VOV-1 was more prevalent in V. destructor than in A. mellifera and we found evidence for viral replication in both hosts. Interestingly, we found differences in viral loads of A. cerana and their V. destructor, A. m. intermissa, and its V. destructor showed partial similarity, while A. m. ligustica and A. m. syriaca and their varroa where very similar. Deformed wing virus exhibited 82.20%, 99.20%, 97.90%, and 0.76% of total viral reads in A. m. ligustica, A. m. syriaca, A. m. intermissa, and A. cerana, respectively. This is the ﬁrst report of a complete segmented-single-stranded negative-sense RNA virus genome in honeybees and V. destructor mites. Keywords: honeybee subspecies; Varroa destructor; metagenomics analysis; viruses Received: 1 November 2018; Accepted: 22 January 2019; Published: 24 January 2019 viruses viruses Article New Viruses from the Ectoparasite Mite Varroa destructor Infesting Apis mellifera and Apis cerana Soﬁa Levin 1,2, Noa Sela 3 , Tal Erez 2, David Nestel 2, Jeffery Pettis 4, Peter Neumann 4 and Nor Chejanovsky 2,* Soﬁa Levin 1,2, Noa Sela 3 , Tal Erez 2, David Nestel 2, Jeffery Pettis 4, Peter Neumann 4 and Nor Chejanovsky 2,* Soﬁa Levin 1,2, Noa Sela 3 , Tal Erez 2, David Nestel 2, Jeffery Pettis 4, Peter Neumann 4 d Ch j k 2 * 1 Department of Entomology, The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot 76100, Israel; whirlingsoﬁa@gmail.com 2 Department of Entomology Institute of Plant Protection, Agricultural Research Organization, Rishon LeZion 7528809, Israel; tal.erez2@mail.huji.ac.il (T.E.); nestel@volcani.agri.gov.il (D.N.) 3 Department of Plant Pathology and Weed Research, Institute of Plant Protection, Agricultural Research Organization, Rishon LeZion 7528809, Israel; noa@volcani.agri.gov.il 4 Institute of Bee Health, Vetsuisse Faculty, University of Bern, 3003 Bern, Switzerland; Pettis.jeff@gmail.com (J.P.); peter.neumann@vetsuisse.unibe.ch (P.N.) * Correspondence: ninar@volcani.agri.gov.il; Tel.: +97239683694 * Correspondence: ninar@volcani.agri.gov.il; Tel.: +97239683694 Viruses 2019, 11, 94 Viruses 2019, 11, 94 Viruses are important pathogens of the honeybee. Different studies reported that Acute bee paralysis virus (ABPV), Deformed wing virus (DWV), Israel acute paralysis virus (IAPV), Kashmir bee virus (KBV), Sacbrood virus (SBV), Slow bee paralysis virus (SBPV), Chronic bee paralysis virus (CBPV), Lake Sinai virus (LSV), Apis mellifera ﬁlamentous virus (AmFV), and Black queen cell virus (BQCV) are pathogenic to honeybees [4,7–11]. p g y Some of the above viruses DWV, SBPV, ABPV, IAPV, and KBV—are transmitted by V. destructor, which has become a growing threat to the existence of A. mellifera [2,3,12–15]. V. destructor and viral pathogens play a key role in the loss of A. mellifera colonies, especially because both the mite and the viruses produce a double effect in colony infestation: V. destructor being tightly linked to honeybee life cycle causes a considerable impact on the brood by feeding on it and transmitting viruses [4,16–18]. Viruses being vectored by mites and thus directly introduced into the pupa’s body during feeding might reach noticeably higher titers in developing honeybees [17,19,20]. Together they cause a higher impact on honeybee colony health and even induce its collapse [12,21,22]. p y y p [ , , ] From an evolutionary point of view, the shift of V. destructor from A. cerana to A. mellifera is a relatively short period of time for the populations of the pathogen and the host to adapt and thus, A. mellifera is a new host for V. destructor. This is valid for its viral content as well. To look for possible variations in virus composition we investigated the viral populations in samples of three A. mellifera subspecies from the Middle East and North Africa (MENA) regions and their parasitic V. destructor mites as well as from A. cerana and their V. destructor mites. For this purpose we used next-generation sequencing (NGS) and performed metagenomics analysis of viral populations from samples of A. m. ligustica colonies from Israel [23] and their V. destructor mites, from Western honeybee subspecies from the MENA region—A. m. intermissa and A. m. syriaca [24]—and their V. destructor mites, and A. cerana and their V. destructor mites from Thailand. In this study, the viral composition and loads from samples of each bee species and its mite were determined, viral loads and viruses of interest were validated and characterized using molecular biology tools. Viruses 2019, 11, 94 This analysis indicated differences in viral composition and load in the samples of the above honeybee subspecies and their parasitizing varroa mites. We found that in these samples A. cerana and their parasite V. destructor differed considerably in their viral load, A. m. intermissa and its varroa mites showed partial similarity in their viral load, while A. m. ligustica and A. m. syriaca and their corresponding varroa mites’ viral loads were more similar. Moreover, besides identifying known and recently discovered viruses, we found two novel viruses—an Orthomyxovirus common to A. mellifera and V. destructor—that we named Varroa orthomyxovirus-1 (VOV-1) and a homolog of the Hubei like-Virga virus 14 that we named VDV-4. VOV-1 showed 23–58% homology to the Orhomyxoviruses Thogoto and Dhori (THOV and DHOV), which bear negative-sense single stranded RNA genomes of six segments [25–28]. Replication of single-stranded sense RNA viruses requires the synthesis of the complementary positive-sense RNA, which can be detected by using strand-speciﬁc RT-PCR [29]. Using this approach we were able to demonstrate replication of VOV-1. This is the ﬁrst report of the presence of a segmented negative-sense ssRNA virus in honeybees and varroa mites. 1. Introduction The mite Varroa destructor is an obligatory ectoparasite of the Eastern honeybee Apis cerana [1]. V. destructor mites spend most of their life cycle inside the colony, reproducing on the honeybee brood and feeding on the pupa. Mites transfer from one host pupa to another on nurse bees that take care of pupa [1]. During the feeding on its host this mite may transfer populations of microorganisms that it bears, and viruses in particular, as well as acquire those that belong to the host [2–5]. V. destructor made a host shift to the Western honeybee A. mellifera at the beginning of the 20th century and spread to Europe, USA, New Zealand, Africa, and the Middle East from southern and southeastern Asia during the last century [6]. Viruses 2019, 11, 94; doi:10.3390/v11020094 www.mdpi.com/journal/viruses 2 of 15 2.2. Samples Preparation RNA extraction from all A. mellifera and mite samples was carried out using TRI Reagent® (Sigma-Aldrich, Israel) according to the manufacturer’s instructions as published before [24,30]. RNA from A. cerana drones and the corresponding V. destructor mites was individually extracted using a GenJet RNA puriﬁcation Kit (Thermo Scientiﬁc, Burlington, Canada) according to the manufacturer’s instructions. 2.4. RT-PCR cDNA was prepared using RevertAid Reverse Transcriptase (Thermo Scientiﬁc) with oligo-dT and random primers according to the manufacturer’s instructions. One-hundred nanogram and 2000 ng RNA templates were used from V. destructor and honeybee samples, respectively. RT-conditions: incubation of RNA and primers at 65 ◦C for 5 min., followed by addition of buffer containing 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 2 mM MgCl2, 5 mM DTT, 4 units of RNase inhibitor Ribolock® (Thermo Scientiﬁc), and the RT enzyme (200 units) in a 25 µL volume, and further incubation at 55 ◦C for 30 min. The reaction was terminated by heating at 85 ◦C for 5 min. PCR-validations were performed with GoTaq® (Promega Corporation, Madison, WI, USA) using 1 µL cDNA template and 0.2 µM of each forward and reverse primer in a 20 µL reaction with the following conditions; 95 ◦C for 4 min, 32 cycles at 94 ◦C for 30 s, then 56 ◦C (VOV-1 segments 1,2,4,5) or 57 ◦C (VOV-1 segments 3,6) for 50 s, 72 ◦C for 2 min. (VOV-1 segments 1,2,4,5) or 1 min. (VOV-1 segments 3,6), and a ﬁnal extension step of 72 ◦C for 10 min. For VDV-4 the PCR conditions were identical to those used in segments 1, 2, 4, 5 of VOV-1 with the corresponding speciﬁc primers. Speciﬁc primers used for validations are described in the Tables S1 and S2 in the Supplementary material. 2.5. VOV-1 Prevalence VOV-1 prevalence was determined by using RT-PCR to detect the presence of the segment 6 of the virus genome in samples of V. destructor mites and honeybees in apiaries located at the North (Haifa, Kibbutz Lehavot HaBashan, Kibbutz Dan), the Center (ARO, Nitzanei Oz, Herut, Kfar Ruth) and the South (Kibbutz Yad Mordechai) of Israel. 2.1. Sample Collection The experimental colonies of A. m. ligustica (worker bees N = 48, from hives 1, 3, 5, and 23, (4 bees per hive), 14 (6 bees), 81 (9 bees), and 401 (1 bee); mites N = 606, from the same hives (85 of them from emerging bees and the rest from free falling mites), collected between October to February 2016, were described before [23]; A. m. syriaca (workers N = 15, from a subcollection of 500 workers from colonies from several apiaries; mites = 20, from tens of mites that were collected in 2013) and A. m. intermissa (workers N = 15, from a subcollection of 500 workers; V. destructor mites N = 27, from tens of mites that were collected in 2013) were described previously [24]. A. cerana drones (N = 6) and V. destructor mites (N = 20) were sampled during spring 2016 (December) in Phrae, Thailand from two colonies 3c 3 of 15 Viruses 2019, 11, 94 and 4c untreated against mites (3 drones and 20 mites from each colony, respectively). The honeybees and corresponding mites were transported in RNA later™and stored immediately at −80 ◦C until RNA extraction. 2.3. Transcriptome and Virome Analysis Construction and paired-end sequencing of the libraries from A. cerana and its corresponding V. destructor mites-RNA samples was performed at the Technion Genome Center on a HiSEq 2000 platform (Illumina, Haifa, Israel). Paired-end reads were assembled de novo using Trinity [31]. The obtained contigs were translated and aligned to the GenBank nonredundant (NR) database by Blastx [32]. Next-generation sequencing (NGS) of the RNA from A. m. ligustica, A. m. intermissa, and A. m. syriaca honeybees and corresponding V. destructor mites was described previously [23,24]. Metagenomic analysis of A. m. ligustica, A. m. intermissa, and A. m. syriaca subspecies and A. cerana bees, and of V. destructor mites samples were carried out as described previously and in Section 2.8 [30]. 2.7. Replication Assay Testing for viral replication (presence of the positive strand-sense RNA) was performed by synthetizing the negative-strand cDNA of fragment 6 from the RNA samples using the tagged primer VOV6-46F-TAG, as we described before to analyze replication of BRV-1 [30]. Subsequently the residual VOV6-46F-TAG primer was inactivated by adding to the mixture exonuclease-I and incubating it for another 15 min. at 37 ◦C (method described in de Miranda et al, 2013 [33]). Finally the exonuclease I was inactivated by heating the mixture at 80 ◦C for 15 min. Subsequently, PCR was performed with primers VOV6-870R and TAG (Supplementary material 1 Table S1). cDNA produced without any primer was used as control in the same reactions followed by PCR with the same primers as above. PCR was performed at 95 ◦C for 4 min., 30 cycles at 94 ◦C for 30 s, then 58 ◦C for 50 s, 72 ◦C for 1 min. and a ﬁnal extension step of 72 ◦C for 10 min. The identity of the ampliﬁed fragment was conﬁrmed by Sanger sequencing (performed at the Biological Services Unit of the Weizmann Institute of Science, Israel). 2.6. qRT-PCR Viral genome copy number was quantiﬁed on a PikoReal 96 machine (Thermo Scientiﬁc) using a standard protocol (95 ◦C 2 min; 40 cycles of 95 ◦C 10 s, 60 ◦C 20 s, 72 ◦C 20 s). Each quantitative PCR analysis was performed in triplicate. Nontemplate controls (water) were included in triplicates in each assay. The KAPA SYBR FAST qPCR Master Mix (2×) Universal (Kapa Bio-systems) was used, in a 10 µL ﬁnal volume. For each analysis 2 µL of the diluted cDNA was used (dilution factor of 4) 4 of 15 Viruses 2019, 11, 94 and speciﬁc primers VOV-1-qRT-F1 and VOV-1-qRT-R1 at a concentration of 0.25 µM each (Table S1). The speciﬁcity of the amplicons synthesized during the PCR run was ascertained by performing a dissociation curve protocol from 60 ◦C to 95 ◦C. Speciﬁc primers used for quantiﬁcation are provided in Table S1 in Supplementary material. 3. Results 3.1. Metagenomic Analysis of Viruses in A. mellifera, A. cerana and Their V. destructor Mites 3.1. Metagenomic Analysis of Viruses in A. mellifera, A. cerana and Their V. destructor Mites In the analysis we included libraries from A. m. ligustica, A. m. syriaca, A. m. intermissa, A. cerana, and their V. destructor counterparts (see Materials and Methods). This revealed variation in composition of the viral loads of A. m. ligustica, A. m. syriaca, A. m. intermissa, A. cerana, and their V. destructor mite counterparts (libraries IB1, SB2, AB3, BCER, IV4, SV5, AV6, and VCER, respectively). Mapping of the libraries’ reads to viral contigs of honeybees and V. destructor mites resulted in In the analysis we included libraries from A. m. ligustica, A. m. syriaca, A. m. intermissa, A. cerana, and their V. destructor counterparts (see Materials and Methods). This revealed variation in composition of the viral loads of A. m. ligustica, A. m. syriaca, A. m. intermissa, A. cerana, and their V. destructor mite counterparts (libraries IB1, SB2, AB3, BCER, IV4, SV5, AV6, and VCER, respectively). Mapping of the libraries’ reads to viral contigs of honeybees and V. destructor mites resulted in 1. identiﬁcation of the most common honeybee viruses: Acute bee paralysis virus (ABPV), Israeli acute paralysis virus (IAPV) in IB1, IV4, and SB2; Apis mellifera ﬁlamentous virus (AmFV) in AB3; Bee Macula-like virus/Varroa Macula-like virus (BeeMLV/VdMLV) and Black queen cell virus (BQCV) in all the libraries except for BCER and VCER; Deformed wing virus (DWV) in all the libraries; Lake Sinai virus (LSV) in IB1; and Sacbrood virus (SBV) in IB1, IV4, SV5, and AV6 (details are provided in Materials and Methods and in Table 3). 2. identiﬁcation of recently described viruses Apis rhabdovirus-1/Bee rhabdovirus-1(ARV-1/ BRV-1) [30], Varroa destructor virus-2 (VDV-2), and Varroa destructor virus-3 (VDV-3) [23]. 3. discovery of two new viruses that we designed Varroa orthomyxovirus-1 (VOV-1), an orthomyxovirus with low homology to other viruses from the Orthomyxoviridae family, and Varroa destructor virus-4 VDV-4 (Table 3 and see below). Table 3. Presence of viruses in honeybees and V. destructor mites by library. A. mellifera ligustica A. mellifera syriaca A. mellifera intermissa A. 2.8. Bioinformatic Identiﬁcation of Contigs Each RNAseq library was de novo-assembled using Trinity assembler version 2.2.0 [31]. The assembled contigs were then searched with BLASTX [33] against the NCBI nonredundant protein database (NR) [34]. After the assembled viruses were identiﬁed in each library, each library’s raw data reads were mapped using bowtie2 [35] to evaluate the virus quantity in the transcriptome. We reanalyzed the data obtained before in A. m. ligustica [23] and realized that contigs of a new Orthomyxovirus were present. Then we analyzed data that we downloaded from recently published transcriptomes of viruses of A. m. intermissa and A. m. syriaca and their varroa from the MENA region [24] and were able to assemble the complete genome of the virus (Tables 1 and 2). Table 1. Libraries used in this study. Table 1. Libraries used in this study. NGS Libraries Library Code Accession Number * A. m. ligustica IB1 PRJNA329428 A. m. syriaca SB2 PRJNA437728 A. m. intermissa AB3 PRJNA437730 A. cerana BCER PRJNA475853 V. destructor from A. m. ligustica IV4 PRJNA329427 V. destructor from A. m. syriaca SV5 PRJNA437729 V. destructor from A. m. intermissa AV6 PRJNA437731 V. destructor from A. cerana VCER PRJNA475855 * Libraries uploaded NCBI short read archive database, raw data. Table 1. Libraries used in this study. Table 2. Accession numbers for the new viruses. Virus Accession Numbers Varroa orthomyxovirus-1 (VOV-1) MK032465 MK032466 MK032467 MK032468 MK032469 MK032470 Varroa destructor virus-4 MK032464 Table 2. Accession numbers for the new viruses. 5 of 15 Viruses 2019, 11, 94 Viruses 2019, 11, 94 2.9. Molecular Phylogenetic Analysis 2.9. Molecular Phylogenetic Analysis Phylogenetic analysis was done using MEGA 6 [34]. Alignment of the proteins was done using MAFFT [36], and then Maximum likelihood Phyml 3.0 was used for creating the tree [37] with a 100 bootstrap. 3. Results cerana libraries—BCER and VCER—were distinct (Figure 1 and see below). For instance, AV6 showed a smaller percentage of DWV reads compared to AB3 and to the other A. mellifera—IB1 and SB2—and their corresponding V. destructor libraries—IV4 and SV5, respectively (Figure 1). The cDNA libraries of A. cerana (BCER) and its varroa (VCER) displayed extremely low percentage of viral reads for DWV (0.7601% and 0.6402%, accordingly). As can be seen, our samples BCER and VCER differed in their load of other viruses as well; while the BCER main viral component was ARV-2 [38], the main virus present in VCER was VDV-2 (Figure 1). In addition, VCER displayed two viruses present in varroa parasites of A. cerana only with 38% homology to Hubei picorna-like virus 29 [39] and 36% homology to Hubei virga-like virus 14 [40] (Table 3 and see below). g The presence of viruses varied across A. mellifera and A. cerana bees and their corresponding V. destructor libraries. From the A. mellifera libraries analyzed, IB1 and IV4 (A. m. ligustica) showed a similar percentage of viral reads of DWV, as did SB2 and SV5 (A. m. syriaca) and A. m. intermissa, and their V. destructor libraries—AB3 and AV6—showed large differences (Figure 1). Also, A. cerana libraries—BCER and VCER—were distinct (Figure 1 and see below). For instance, AV6 showed a smaller percentage of DWV reads compared to AB3 and to the other A. mellifera—IB1 and SB2—and their corresponding V. destructor libraries—IV4 and SV5, respectively (Figure 1). The cDNA libraries of A. cerana (BCER) and its varroa (VCER) displayed extremely low percentage of viral reads for DWV (0.7601% and 0.6402%, accordingly). As can be seen, our samples BCER and VCER differed in their load of other viruses as well; while the BCER main viral component was ARV-2 [38], the main virus present in VCER was VDV-2 (Figure 1). In addition, VCER displayed two viruses present in varroa parasites of A. cerana only with 38% homology to Hubei picorna-like virus 29 [39] and 36% homology t H b i i lik i 14 [40] (T bl 3 d b l ) g [ ] ( ) Figure 1. Presence of viruses in honeybee and V. destructor mite by library. Percentage of total viral reads* mapping to viral contigs of the honeybees and V. destructor mites libraries. 3. Results cerana Library Virus IB1 IV4 SB2 SV5 AB3 AV6 BCER VCER Acute bee paralysis virus (ABPV+IAPV) + + + Aphis glycines virus-1 (ApGlV1) + Apis mellifera ﬁlamentous virus (AmFV) + Apis rhabdovirus-1/Bee rhabdovirus (ARV-1/BRV-1) + + + + + + Apis rhabdovirus-2 (ARV-2) + + + + + + Bee/Varroa destructor Macula-like virus (BeeMLV/ VdMLV) + + + + + + + Beihai horseshoe crab virus-1 + Black queen cell virus (BQCV) + + + + + + Cyclovirus + + + Deformed wing virus (DWV) + + + + + + + + Varroa orthomyxovirus-1 (VOV-1) + + + + Hubei picorna-like virus-29 + Hubei virga-like virus-14 (Varroa destructor virus 4, VDV-4) + Lake Sinai virus (LSV) + Sacbrood virus (SBV) + + + + Varroa destructor virus-2 (VDV-2) + + + + Varroa destructor virus-3 (VDV-3) + + + Determined from the percentage of total viral reads mapping to viral contigs of the honeybees (IB1, SB2, AB3, and BCER) and V. destructor mites (IV4, SV5, AV6, and VCER) libraries with a cutoff at 0.0001%. BLASTX against Genbank, NCBI. Table 3. Presence of viruses in honeybees and V. destructor mites by library. Determined from the percentage of total viral reads mapping to viral contigs of the honeybees (IB1, SB2, AB3, and BCER) and V. destructor mites (IV4, SV5, AV6, and VCER) libraries with a cutoff at 0.0001%. BLASTX against Genbank, NCBI. Interestingly, ARV-1/BRV-1 and VOV-1 showed differences in their distribution across bee and V. destructor libraries; while ARV-1/BRV-1 was present in all V. destructor libraries and in two of the 6 of 15 6 of 16 Viruses 2019, 11, 94 Viruses 2018, 10, x FO honeybee libraries, VOV-1 was limited to A. m. ligustica and A. m. syriaca and their corresponding varroa (Table 3). Determined from the percentage of total viral reads mapping to viral contigs of the honeybees (IB1, SB2, AB3, and BCER) and V. destructor mites (IV4, SV5, AV6, and VCER) libraries with a cutoff at 0.0001%. BLASTX against Genbank, NCBI. The presence of viruses varied across A. mellifera and A. cerana bees and their corresponding V. destructor libraries. From the A. mellifera libraries analyzed, IB1 and IV4 (A. m. ligustica) showed a similar percentage of viral reads of DWV, as did SB2 and SV5 (A. m. syriaca) and A. m. intermissa, and their V. destructor libraries—AB3 and AV6—showed large differences (Figure 1). Also, A. 3 2 Varroa Orthomyxovirus 1 and the Hubei Virga like 14 Homolog Virus 3.2. Varroa Orthomyxovirus-1 and the Hubei Virga-like 14 Homolog Virus 3.2. Varroa Orthomyxovirus 1 and the Hubei Virga like 14 Homolog Virus We reanalyzed the data obtained before in A. m. ligustica [23] and realized that contigs of a new Orthomyxovirus were present in the data. To complete the picture we downloaded and analyzed data from the transcriptome of viruses of A. m. intermissa and A. m. syriaca and their varroa mites from the Middle East and North African (MENA) honeybees and varroa mites that were published recently but did not focus on finding new viruses [24]. This additional sequence data facilitated complete genome assembly of this new virus. According to BLASTX analysis we identified VOV-1 contigs in two cDNA libraries of bees (IB1 and SB2) and two cDNA libraries of their corresponding varroa mites (IV4 and SV5). VOV-1 showed 23–58% homology to the Orthomyxoviruses Thogoto and Dhori (THOV and DHOV), which bear negative-sense single stranded RNA genomes of six segments [25–28]. Contigs of 2198, 1899, and 358 nucleotides in length from the SV5 library showed homology of 58%, 29%, and 46% to polymerase subunits PB2, PB1, and PA encoded in segments 1, 2, and 3 of the DHOV genome, respectively (Table 4). A contig of 232 nucleotides length from the IV4 library showed homology of 41% to the glycoprotein subunit (GP) encoded in segment 4 of THOV; a contig We reanalyzed the data obtained before in A. m. ligustica [23] and realized that contigs of a new Orthomyxovirus were present in the data. To complete the picture we downloaded and analyzed data from the transcriptome of viruses of A. m. intermissa and A. m. syriaca and their varroa mites from the Middle East and North African (MENA) honeybees and varroa mites that were published recently but did not focus on ﬁnding new viruses [24]. This additional sequence data facilitated complete genome assembly of this new virus. According to BLASTX analysis we identiﬁed VOV-1 contigs in two cDNA libraries of bees (IB1 and SB2) and two cDNA libraries of their corresponding varroa mites (IV4 and SV5). VOV-1 showed 23–58% homology to the Orthomyxoviruses Thogoto and Dhori (THOV and DHOV), which bear negative-sense single stranded RNA genomes of six segments [25–28]. 3. Results Including: DWV and genetic variants, ARV-1, ARV-2, VOV-1, VDV-2, VDV-3, and other viruses (11 more, see Table 1), cutoff at 0.0001%. Figure 1. Presence of viruses in honeybee and V. destructor mite by library. Percentage of total viral reads mapping to viral contigs of the honeybees and V. destructor mites libraries. Including: DWV and genetic variants, ARV-1, ARV-2, VOV-1, VDV-2, VDV-3, and other viruses (11 more, see Table 1), cutoff at 0.0001%. Figure 1. Presence of viruses in honeybee and V. destructor mite by library. Percentage of total viral reads mapping to viral contigs of the honeybees and V. destructor mites libraries. Including: DWV and genetic variants, ARV-1, ARV-2, VOV-1, VDV-2, VDV-3, and other viruses (11 more, see Table 1), cutoff at 0.0001%. Figure 1. Presence of viruses in honeybee and V. destructor mite by library. Percentage of total viral reads mapping to viral contigs of the honeybees and V. destructor mites libraries. *Including: DWV and genetic variants, ARV-1, ARV-2, VOV-1, VDV-2, VDV-3, and other viruses (11 more, see Table 1), cutoff at 0.0001%. 3 2 Varroa Orthomyxovirus 1 and the Hubei Virga like 14 Homolog Virus 3.2. Varroa Orthomyxovirus-1 and the Hubei Virga-like 14 Homolog Virus Contigs of 2198, 1899, and 358 nucleotides in length from the SV5 library showed homology of 58%, 29%, and 46% to polymerase subunits PB2, PB1, and PA encoded in segments 1, 2, and 3 of the DHOV genome, respectively (Table 4). A contig of 232 nucleotides length from the IV4 library showed homology of 41% to the glycoprotein subunit (GP) encoded in segment 4 of THOV; a contig of 1442 nucleotides length 7 of 15 Viruses 2019, 11, 94 0, x FOR PEER REVIE Viruses 2019, 11, 94 0, x FOR PEER REVI from IB1 library showed homology of 39% to the nucleoprotein subunit (NP) encoded in segment 5 of DHOV; and a contig of 983 nucleotides from the SB2 library showed homology of 23% to matrix protein (M) encoded in segment 6 of THOV (Table 4). g g y of 23% to matrix protein (M) encoded in segment 6 of THOV (Table 4). 4. Comparison of the Varroa orthomyxovirus-1 (VOV-1) contigs to the Orthomyxoviruses to and Dhori (THOV and DHOV) from IB1 library showed homology of 39% to the nucleoprotein subunit (NP) encoded in segment 5 of DHOV; and a contig of 983 nucleotides from the SB2 library showed homology of 23% to matrix protein (M) encoded in segment 6 of THOV (Table 4). g g y of 23% to matrix protein (M) encoded in segment 6 of THOV (Table 4). 4. Comparison of the Varroa orthomyxovirus-1 (VOV-1) contigs to the Orthomyxoviruses to and Dhori (THOV and DHOV) Table 4. Comparison of the Varroa orthomyxovirus-1 (VOV-1) contigs to the Orthomyxoviruses Thogoto and Dhori (THOV and DHOV). VOV-1 Segments Length of Contig (na) Similarity to THOV/DHOV THOV/DHOV Proteins 1 2198 58% PB2 (DHOV) 2 1899 29% PB1 (DHOV) 3 358 46% PA (DHOV) 4 232 41% GP (THOV) 5 1442 39% NP (DHOV) 6 983 23% M (THOV) ( ) VOV-1 segments Length of contig (na) Similarity to THOV/ DHOV THOV/ DHOV proteins 1 2198 58% PB2 (DHOV) 2 1899 29% PB1 (DHOV) 3 358 46% PA (DHOV) 4 232 41% GP (THOV) 5 1442 39% NP (DHOV) Table 4. Comparison of the Varroa orthomyxovirus-1 (VOV-1) contigs to the Orthomyxoviruses Thogoto and Dhori (THOV and DHOV). Viruses 2019, 11, 94 destructor mite virome: lanes 1, 4, 7, 10, 13, and 16; Honeybee virome: lanes 2, 5, 8, 11, 14, and 17; NTC (nontemplate control): lanes 3, 6, 9, 12, 15, and 18; M1 and M2: GeneRuler MarkerTM 1 kb, 100 bp DNA ladders, respectively. Arrows: VOV-1 segments (1–6). adde s, espective y. A ows: VOV seg e ts ( 6). Furthermore, we tested the presence of segment 6 of the viral genome to estimate the prevalence of VOV-1 by RT-PCR in individual mites and honeybees. We detected VOV-1 in 35.56% of V. destructor mites from Israeli colonies located in ARO, Beit Dagan (16 of 45), and in none of 32 individual honeybees sampled from the same colonies. We also analyzed its prevalence in colonies located in the Center, North, and South of Israel by testing pools of the honeybees and V. destructor mites with the same PCR method. The virus was identified in 78.57% of V. destructor pools (11 of 14 Furthermore, we tested the presence of segment 6 of the viral genome to estimate the prevalence of VOV-1 by RT-PCR in individual mites and honeybees. We detected VOV-1 in 35.56% of V. destructor mites from Israeli colonies located in ARO, Beit Dagan (16 of 45), and in none of 32 individual honeybees sampled from the same colonies. We also analyzed its prevalence in colonies located in the Center, North, and South of Israel by testing pools of the honeybees and V. destructor mites with the same PCR method. The virus was identiﬁed in 78.57% of V. destructor pools (11 of 14 pools) and only in 8.33% of honeybee pools (5 of 60 pools). pools) and only in 8.33% of honeybee pools (5 of 60 pools). The number of genomic copies of VOV-1 estimated by qRT-PCR was similar in individual mites collected from honeybee colonies in ARO, Beit Dagan, in pools of mites, and in honeybee pools sampled from colonies located in the Center, North, and South of Israel: 5.11×102–1.22×106, 2.33×103– The number of genomic copies of VOV-1 estimated by qRT-PCR was similar in individual mites collected from honeybee colonies in ARO, Beit Dagan, in pools of mites, and in honeybee pools sampled from colonies located in the Center, North, and South of Israel: 5.11 × 102–1.22 × 106, 2.33 × 103–4.88 × 105, and 4.91 × 102–1.38 × 105, respectively (Table 5). 4.88×105, and 4.91×102–1.38×105, respectively (Table 5). Viruses 2019, 11, 94 Viruses 2019, 11, 94 Based on the above contigs’ sequences we designed speciﬁc primers to validate the presence of each segment of VOV-1 in the viromes of Israeli A. mellifera ligustica and their counterpart V. destructor parasites (Figure 3 and see Materials and Methods). All six segments were identiﬁed in the V. destructor virome (Figure 3, lanes 1, 4, 7, 10, 13, and 16) but they were absent in the virome of honeybees (Figure 3, lanes 2, 5, 8, 11, 14, and 17). Based on the above contigs' sequences we designed specific primers to validate the presence of each segment of VOV-1 in the viromes of Israeli A. mellifera ligustica and their counterpart V. destructor parasites (Figure 3 and see Materials and Methods). All six segments were identified in the V. destructor virome (Figure 3, lanes 1, 4, 7, 10, 13, and 16) but they were absent in the virome of honeybees (Figure 3, lanes 2, 5, 8, 11, 14, and 17). Figure 3. Presence of VOV-1 in virome of V. destructor mites and its absence in honeybees. V. destructor mite virome: lanes 1, 4, 7, 10, 13, and 16; Honeybee virome: lanes 2, 5, 8, 11, 14, and 17; NTC (nontemplate control): lanes 3, 6, 9, 12, 15, and 18; M1 and M2: GeneRuler MarkerTM 1 kb, 100 bp DNA l dd i l A VOV 1 (1 6) Figure 3. Presence of VOV-1 in virome of V. destructor mites and its absence in honeybees. V. destructor mite virome: lanes 1, 4, 7, 10, 13, and 16; Honeybee virome: lanes 2, 5, 8, 11, 14, and 17; NTC (nontemplate control): lanes 3, 6, 9, 12, 15, and 18; M1 and M2: GeneRuler MarkerTM 1 kb, 100 bp DNA ladders, respectively. Arrows: VOV-1 segments (1–6). Figure 3. Presence of VOV-1 in virome of V. destructor mites and its absence in honeybees. V. destructor mite virome: lanes 1, 4, 7, 10, 13, and 16; Honeybee virome: lanes 2, 5, 8, 11, 14, and 17; NTC (nontemplate control): lanes 3, 6, 9, 12, 15, and 18; M1 and M2: GeneRuler MarkerTM 1 kb, 100 bp DNA Figure 3. Presence of VOV-1 in virome of V. destructor mites and its absence in honeybees. V. Viruses 2019, 11, 94 Table 5. Genomic copies of VOV-1 in individual mites and in pools of mites and nurse honeybees. location hive # Sample type N VOV-1 genomic copies ARO (C) 7 Mite 1 3.54 × 105 ARO (C) 13 Mite 1 1.22 × 106 ARO (C) 19 Mite 1 4.43 × 105 ARO (C) 81 Mite 1 9.40 × 104 ARO (C) 7 Mite 1 5.34 × 105 To investigate if VOV-1 replicates in our samples, we screened for presence of the positive-sense RNA strand of the fragment 6 of the virus using RNA-strand sense-speciﬁc primer-tagged RT-PCR (see Materials and Methods). A predicted size fragment of ~840 nucleotides corresponding to the VOV-1 positive-sense-strand RNA between nucleotides 46 and 870 was found in tested samples from individual V. destructor mites (Figure 4, panel A, lanes 3, 9, 11, and 13), or from V. destructor mite and nurse honeybee pools (Figure 4, panels B and C, lanes 15, 17, 19, 21, 23, and 27 and 33, 37, and 39, respectively). No ampliﬁcation was observed in some varroa individuals, varroa and nurse honeybee pools (Figure 4, panels A, B and C, lanes 1, 5, and 7 and 25, 29, 31, and 35, respectively). Control samples obtained when PCR was performed with cDNA prepared from the same RNA without the corresponding oligonucleotide primer in the RT reaction did not show any amplicon (Figure 4, panels A, B, and C, lanes 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, and 38, respectively). ARO (C) 401 Mite 1 6.32 × 105 ARO (C) 401 Mite 1 2.50 × 105 p y We conﬁrmed by Sanger DNA sequencing that the above speciﬁc-primer-tagged amplicons were identical to the VOV-1 sequence comprising nucleotides 46 and 870 of segment 6 of the viral genome. ARO (C) 401 Mite 1 6.32 × 105 ARO (C) 401 Mite 1 2.50 × 105 We conﬁrmed by Sanger DNA sequencing that the above speciﬁc-primer-tagged amplicons were identical to the VOV-1 sequence comprising nucleotides 46 and 870 of segment 6 of the viral genome. 3 2 Varroa Orthomyxovirus 1 and the Hubei Virga like 14 Homolog Virus 3.2. Varroa Orthomyxovirus-1 and the Hubei Virga-like 14 Homolog Virus VOV-1 Length of contig Similarity to THOV/ THOV/ DHOV Phylogenetic analysis of open reading frames (ORFs) coding for polymerase subunits PB2, PB1, and PA showed that the polymerase was closely related to negative-sense ssRNA viruses belonging to the Orthomyxoviridae viral family: Thogoto virus (THOV); Aransas Bay virus (ABV); Upolu virus (UPOV) (Figure 2A–C); and Jos virus (JOSV) (Figure 2A,C). PB2 is phylogenetically closer to THOV, ABV, UPOV, and JOSV, and more distant from DHOV and Bourbon virus (BRBV) as well as PB1, except for JOSV, and PA, except for BRBV (Figure 2A–C). 6 983 23% M (THOV) genetic analysis of open reading frames (ORFs) coding for polymerase subunits PB2, PB1, owed that the polymerase was closely related to negative-sense ssRNA viruses belonging omyxoviridae viral family: Thogoto virus (THOV); Aransas Bay virus (ABV); Upolu virus igure 2A–C); and Jos virus (JOSV) (Figure 2A,C). PB2 is phylogenetically closer to THOV, V, and JOSV, and more distant from DHOV and Bourbon virus (BRBV) as well as PB1, JOSV, and PA, except for BRBV (Figure 2A–C). Maximum-likelihood phylogenetic tree of VOV-1 contigs for segments 1, 2, and 3. The trees ructed based on the ORF of VOV-1 polymerase subunits PB2 (A), PB1 (B), and PA (C). accession numbers provided. Figure 2. Maximum-likelihood phylogenetic tree of VOV-1 contigs for segments 1, 2, and 3. The trees were constructed based on the ORF of VOV-1 polymerase subunits PB2 (A), PB1 (B), and PA (C). GeneBank accession numbers provided. ximum-likelihood phylogenetic tree of VOV-1 contigs for segments 1, 2, and 3. The trees ucted based on the ORF of VOV-1 polymerase subunits PB2 (A), PB1 (B), and PA (C). ccession numbers provided. Figure 2. Maximum-likelihood phylogenetic tree of VOV-1 contigs for segments 1, 2, and 3. The trees were constructed based on the ORF of VOV-1 polymerase subunits PB2 (A), PB1 (B), and PA (C). GeneBank accession numbers provided. 8 of 15 Viruses 2019, 11, 94 Viruses 2019, 11, 94 ARO (C) 6 Mite 1 4.23 × 105 ARO (C) 7 Mite 1 2.69 × 105 ARO (C) 13 Mite 1 4.00 × 105 ARO (C) 13 Mite 1 1.83 × 104 ARO (C) 19 Mite 1 4.62 × 105 From the two undescribed viruses that we found in VCER we further investigated VDV-4. Phylogenetic analysis using the putative large ORF protein of the virus showed that it is 36% homologous to the hypothetical protein gene of spider viruses Hubei virga- like virus 14 and Hubei virga-like virus 13 as well as to the spider putative protein of the virus Nephila clavipes virus 4 (Figure 5). 9 of 15 f 16 Viruses 2019, 11, 94 Viruses 2018, 1 Table 5. Genomic copies of VOV-1 in individual mites and in pools of mites and nurse honeybees. Viruses 2019, 11, 94 Location Hive # Sample Type N VOV-1 Genomic Copies ARO (C) 7 Mite 1 3.54 × 105 ARO (C) 13 Mite 1 1.22 × 106 ARO (C) 19 Mite 1 4.43 × 105 ARO (C) 81 Mite 1 9.40 × 104 ARO (C) 7 Mite 1 5.34 × 105 ARO (C) 401 Mite 1 6.32 × 105 ARO (C) 401 Mite 1 2.50 × 105 ARO (C) 6 Mite 1 4.23 × 105 ARO (C) 7 Mite 1 2.69 × 105 ARO (C) 13 Mite 1 4.00 × 105 ARO (C) 13 Mite 1 1.83 × 104 ARO (C) 19 Mite 1 4.62 × 105 ARO (C) 19 Mites 1 5.11 × 102 ARO (C) 11 Mites 1 4.19 × 105 ARO (C) 9 Mites (p) 6 3.27 × 104 ARO (C) 10 Mites (p) 6 2.85 × 104 ARO (C) 7 Mites (p) 6 2.95 × 104 ARO (C) 38 Mites (p) 6 2.01 × 104 ARO (C) 401 Mites (p) 6 1.44 × 104 Nitzanei Oz (C) 1 Mites(p) 6 2.73 × 104 Haifa (N) 1 Mite (p) 6 1.35 × 104 Lehavot Habashan (N) 1 Mite (p) 6 1.64 × 105 Lehavot Habashan (N) 2 Mites (p) 6 4.88 × 105 Lehavot Habashan (N) 4 Mites (p) 6 2.33 × 103 ARO (C) MIX Mites (p) 6 3.08 × 105 Kfar Rut (C) 17 Bees (p) 10 2.01 × 104 Dan (N) 14 Bees (p) 10 4.91 × 102 Kfar Rut (S) 43 Bees (p) 10 1.38 × 105 Yad Mordehai (S) 13 Bees (p) 10 1.25 × 103 Yad Mordehai (S) 36 Bees (p) 10 9.99 × 104 Quantitation of VOV-1 genomic copy number was carried out by amplifying segment 6 using speciﬁc primers (see Materials and Methods). N = North, C = Center, and S = South of Israel, respectively. MIX, group of mites from various colonies. (p), pool. Viruses 2019, 11, 94 ( ) (p) ARO (C) 10 Mites (p) 6 2.85 × 104 Quantitation of VOV-1 genomic copy number was carried out by amplifying segment 6 using speciﬁc primers (see Materials and Methods). N = North, C = Center, and S = South of Israel, respectively. MIX, group of mites from various colonies. (p), pool. pools (Figure 4, panels A, B and C, lanes 1, 5, and 7 and 25, 29, 31, and 35, respectively). Control samples obtained when PCR was performed with cDNA prepared from the same RNA without the corresponding oligonucleotide primer in the RT reaction did not show any amplicon (Figure 4, panels A, B, and C, lanes 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, and 38, respectively). Figure 4. Detection of the VOV-1-segment 6 positive-sense RNA strand in V. destructor and A. mellifera. (A) V. destructor individuals, odd sample numbers and their respective controls, even sample numbers. (B) V. destructor (pools) odd samples, and their respective controls, even samples. (C) Lanes Figure 4. Detection of the VOV-1-segment 6 positive-sense RNA strand in V. destructor and A. mellifera. (A) V. destructor individuals, odd sample numbers and their respective controls, even sample numbers. (B) V. destructor (pools) odd samples, and their respective controls, even samples. (C) Lanes 29 and 30, V. destructor (pool) and its respective control; A. mellifera (pools), odd lanes 31–39 and respective controls, even lanes 32–40. PCR control reaction of the same individual RNA performed on cDNA produced without any primer (see the section “Materials and Methods”. PCR primers: VOV6-870R and TAG. M, GeneRuler Marker 1 kb DNA Ladder (Thermo Scientiﬁc Inc.); 41 and 42: nontemplate control; Arrow, VOV-1 amplicon that was conﬁrmed by sequencing (see “Materials and Methods”). C, lanes 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, and 38, respect Figure 4. Detection of the VOV-1-segment 6 positive-sense RNA strand in V. destructor and A. mellifera. (A) V. destructor individuals, odd sample numbers and their respective controls, even sample numbers. (B) V. destructor (pools) odd samples, and their respective controls, even samples. (C) Lanes Figure 4. Detection of the VOV-1-segment 6 positive-sense RNA strand in V. destructor and A. mellifera. (A) V. destructor individuals, odd sample numbers and their respective controls, even sample numbers. (B) V. Viruses 2019, 11, 94 ARO (C) 9 Mites (p) 6 3.27 10 ARO (C) 10 Mites (p) 6 2.85 × 104 ARO (C) 7 Mites (p) 6 2.95 × 104 ARO (C) 38 Mites (p) 6 2.01 × 104 ARO (C) 401 Mites (p) 6 1.44 × 104 Nitzanei Oz (C) 1 Mites(p) 6 2.73 × 104 Haifa (N) 1 Mite (p) 6 1.35 × 104 Lehavot Habashan (N) 1 Mite (p) 6 1.64 × 105 Lehavot Habashan (N) 2 Mites (p) 6 4.88 × 105 Lehavot Habashan (N) 4 Mites (p) 6 2.33 × 103 ARO (C) MIX Mites (p) 6 3.08 × 105 Kfar Rut (C) 17 Bees (p) 10 2.01 × 104 Dan (N) 14 Bees (p) 10 4.91 × 102 Kfar Rut (S) 43 Bees (p) 10 1.38 × 105 Yad Mordehai (S) 13 Bees (p) 10 1.25 × 103 Yad Mordehai (S) 36 Bees (p) 10 9.99 × 104 Quantitation of VOV-1 genomic copy number was carried out by amplifying segment 6 using specific primers (see Materials and Methods). N = North, C = Center, and S = South of Israel, respectively. MIX, group of mites from various colonies. (p), pool. To investigate if VOV-1 replicates in our samples, we screened for presence of the positive-sense RNA strand of the fragment 6 of the virus using RNA-strand sense-specific primer-tagged RT-PCR (see Materials and Methods). A predicted size fragment of ~840 nucleotides corresponding to the VOV-1 positive-sense-strand RNA between nucleotides 46 and 870 was found in tested samples from individual V. destructor mites (Figure 4, panel A, lanes 3, 9, 11, and 13), or from V. destructor mite and nurse honeybee pools (Figure 4, panels B and C, lanes 15, 17, 19, 21, 23, and 27 and 33, 37, and 39, respectively). No amplification was observed in some varroa individuals, varroa and nurse honeybee pools (Figure 4, panels A, B and C, lanes 1, 5, and 7 and 25, 29, 31, and 35, respectively). Control samples obtained when PCR was performed with cDNA prepared from the same RNA without the corresponding oligonucleotide primer in the RT reaction did not show any amplicon (Figure 4, panels Table 5. Genomic copies of VOV-1 in individual mites and in pools of mites and nurse honeybees. Viruses 2019, 11, 94 destructor (pools) odd samples, and their respective controls, even samples. (C) Lanes 29 and 30, V. destructor (pool) and its respective control; A. mellifera (pools), odd lanes 31–39 and respective controls, even lanes 32–40. PCR control reaction of the same individual RNA performed on cDNA produced without any primer (see the section “Materials and Methods”. PCR primers: VOV6-870R and TAG. M, GeneRuler Marker 1 kb DNA Ladder (Thermo Scientiﬁc Inc.); 41 and 42: nontemplate control; Arrow, VOV-1 amplicon that was conﬁrmed by sequencing (see “Materials and Methods”). Figure 4. Detection of the VOV-1-segment 6 positive-sense RNA strand in V. destructor and A. mellifera. (A) V. destructor individuals, odd sample numbers and their respective controls, even sample numbers. (B) V. destructor (pools) odd samples, and their respective controls, even samples. (C) Lanes Figure 4. Detection of the VOV-1-segment 6 positive-sense RNA strand in V. destructor and A. mellifera. (A) V. destructor individuals, odd sample numbers and their respective controls, even sample numbers. (B) V. destructor (pools) odd samples, and their respective controls, even samples. (C) Lanes 29 and 30, V. destructor (pool) and its respective control; A. mellifera (pools), odd lanes 31–39 and respective controls, even lanes 32–40. PCR control reaction of the same individual RNA performed on cDNA produced without any primer (see the section “Materials and Methods”. PCR primers: VOV6-870R and TAG. M, GeneRuler Marker 1 kb DNA Ladder (Thermo Scientiﬁc Inc.); 41 and 42: nontemplate control; Arrow, VOV-1 amplicon that was conﬁrmed by sequencing (see “Materials and Methods”). 10 of 15 s virus 4 Viruses 2019, 11, 94 virga like virus (Figure 5). Figure 5. Genomic organization (A) and maximum-likelihood phylogenetic tree (B) of VDV-4. The t t t d b d th ORF f VDV 4 G B k i b id d Figure 5. Genomic organization (A) and maximum-likelihood phylogenetic tree (B) of VDV-4. The tree was constructed based on the ORF of VDV-4. GeneBank accession numbers provided. Figure 5. Genomic organization (A) and maximum-likelihood phylogenetic tree (B) of VDV-4. The d b d h ORF f VDV 4 G B k i b id d Figure 5. Genomic organization (A) and maximum-likelihood phylogenetic tree (B) of VDV-4. The tree was constructed based on the ORF of VDV-4. GeneBank accession numbers provided. tree was constructed based on the ORF of VDV 4. GeneBank accession numbers provided. Viruses 2019, 11, 94 RT-PCR validation of its presence in a small sample of A. cerana drones and V. destructor individuals suggested that this virus was predominant in the latter (Figure 6). Therefore, we decided RT-PCR validation of its presence in a small sample of A. cerana drones and V. destructor individuals suggested that this virus was predominant in the latter (Figure 6). Therefore, we decided to name it VDV-4. Viruses 2018, 10, x FOR PEER REVIEW 11 of 16 o name it VDV-4. Figure 6. Presence of VDV-4 in V. destructor mites and A. cerana drones: lanes 1–3 and 4–6 pools of 5 V. destructor cDNAs each from colonies 3c and 4c, respectively; lanes 7–9 and 10–12 individual A. cerana drones, 3 per colony; lanes (−) nontemplate control; lanes 1 kb, GeneRuler MarkerTM 1 kb DNA ladder. Arrows: VDV-4, Upper panel VDV-4 genome 5’-end primers VDV4-1451F and VDV4-2889R, expected fragment 1438 bp; lower panel VDV-4 genome 3’-end primers VDV4-6242F and VDV4- 7818R e e ted f a e t 1576 b Figure 6. Presence of VDV-4 in V. destructor mites and A. cerana drones: lanes 1–3 and 4–6 pools of 5 V. destructor cDNAs each from colonies 3c and 4c, respectively; lanes 7–9 and 10–12 individual A. cerana drones, 3 per colony; lanes (−) nontemplate control; lanes 1 kb, GeneRuler MarkerTM 1 kb DNA ladder. Arrows: VDV-4, Upper panel VDV-4 genome 5’-end primers VDV4-1451F and VDV4-2889R, expected fragment 1438 bp; lower panel VDV-4 genome 3’-end primers VDV4-6242F and VDV4-7818R, expected fragment 1576 bp. Figure 6. Presence of VDV-4 in V. destructor mites and A. cerana drones: lanes 1–3 and 4–6 pools of 5 V. destructor cDNAs each from colonies 3c and 4c, respectively; lanes 7–9 and 10–12 individual A. cerana drones, 3 per colony; lanes (−) nontemplate control; lanes 1 kb, GeneRuler MarkerTM 1 kb DNA ladder. Arrows: VDV-4, Upper panel VDV-4 genome 5’-end primers VDV4-1451F and VDV4-2889R, expected fragment 1438 bp; lower panel VDV-4 genome 3’-end primers VDV4-6242F and VDV4- Figure 6. Presence of VDV-4 in V. destructor mites and A. cerana drones: lanes 1–3 and 4–6 pools of 5 V. destructor cDNAs each from colonies 3c and 4c, respectively; lanes 7–9 and 10–12 individual A. cerana drones, 3 per colony; lanes (−) nontemplate control; lanes 1 kb, GeneRuler MarkerTM 1 kb DNA ladder. Viruses 2019, 11, 94 Arrows: VDV-4, Upper panel VDV-4 genome 5’-end primers VDV4-1451F and VDV4-2889R, expected fragment 1438 bp; lower panel VDV-4 genome 3’-end primers VDV4-6242F and VDV4-7818R, expected fragment 1576 bp. 7818R, exp 4. Discussion 4. Discussion We presumed that since Apis mellifera is a new host to Varroa destructor they might show differences both in the composition and distribution of their viral load. Our analysis of the data illustrates changes in viral composition and load among samples from A. mellifera subspecies and their V. destructor. Interestingly, a small-scale sample of A. cerana and its V. destructor showed variation in viral composition and load as well. The variation in virus composition was based on a n = 1 repetition per bee species and V. destructor transcriptomes and, consequently, the results do not We presumed that since Apis mellifera is a new host to Varroa destructor they might show differences both in the composition and distribution of their viral load. Our analysis of the data illustrates changes in viral composition and load among samples from A. mellifera subspecies and their V. destructor. Interestingly, a small-scale sample of A. cerana and its V. destructor showed variation in viral composition and load as well. The variation in virus composition was based on a n = 1 repetition per bee species and V. destructor transcriptomes and, consequently, the results do not necessarily reﬂect variations at the level of species/subspecies and could be due to other factors such as sampling region, season 11 of 15 Viruses 2019, 11, 94 and/or diverse time of sampling of the different bee species and there mites, etc. A higher number of samples in a coordinated effort will be required for species/subspecies comparative purposes. We found that in the libraries studied the viral reads of Deformed wing virus, one of the most important factors affecting honeybee colony health and survival, were 82.20%, 99.20%, and 97.90% in A. m. ligustica, A. m. syriaca, and A. m. intermissa, respectively, and only 0.76% in A. cerana. In addition, we observed that a few viruses of importance were present in A. mellifera libraries but not in A. cerana’s. Namely, ABPV + IAPV in A. m. ligustica and its V. destructor, and in A. m. syriaca; BeeMLV/VdMLV in all A. mellifera libraries and corresponding V. destructor mites, as well as in varroa from A. cerana; BQCV in all A. mellifera and corresponding V. destructor and SBV in A. m. ligustica and all three libraries of varroa mites parasitizing A. mellifera. ABPV seems to be uncommon to A. 7818R, exp 4. Discussion cerana in China and South Korea [41,42], or to show low prevalence in wild colonies [43], as was in A. cerana in Northern Thailand [44]. SBV was reported with high prevalence in Southeast Asia [41,45–47] as well as BQCV, that showed relatively high prevalence in viral populations of A. cerana in China, South Korea, and Vietnam [42,43,48]. These two viruses were absent in our libraries and that could be due also to the small sample size of them. p Furthermore, we identiﬁed recently characterized viruses in A. mellifera and/or in varroa mites, namely Apis rhabdovirus-1/Bee rahbdovirus-1 (ARV-1/BRV-1) [30,38], Varroa destructor virus-2 and -3 (VDV-2 and VDV-3) [23], and a new Varroa orthomyxovirus-1 (VOV-1). Some of these viruses were identiﬁed as common to honeybees and V. destructor like ARV-1/BRV-1 and VOV-1, while others appeared to be restricted to mites, such as VDV-2 and VDV-3. Interestingly, VOV-1’s presence was limited to A. m. ligustica and A. m. syriaca as well as to V. destructor mites parasitizing them. VDV-2 was detected in all V. destructor libraries and VDV-3 was absent in Varroa destructor mites parasitizing A. cerana. ARV-1/BRV-1 was found previously in A. mellifera, V. destructor, and in Bombus impatiens, but our ﬁnding that it is present in A. cerana suggests that it may have a broader host range. V. destructor mite parasites of A. cerana appear to bear two novel viruses with low homology to Hubei picorna-like virus-29 [39] and to Hubei virga-like virus-14 [40], which were absent in V. destructor mites from A. mellifera. We validated the presence of a Hubei virga-like virus-14 and designed it VDV-4. Data suggest that following V. destructor invasion there is high selection on DWV strains such that only a single strain seems to dominate, though which strain dominates varies across colonies and studies [20,49–51]. This process is accompanied with increase in the collapse of V. destructor-infested colonies [15,51,52]. Moreover, laboratory experiments showed that DWV undergoes rapid selection following its injection in the honeybee hemolymph, similarly to what happens during parasitization of Varroa destructor on A. mellifera [20]. In our study, we measured differences in DWV loads between samples of A. mellifera subspecies and their V. destructor counterparts. V. destructor from A. m. intermissa showed lower DWV levels compared to its parasitized host (26.83% and 97.90%, respectively). Interestingly, it was reported that A. m. intermissa was more resistant to V. destructor parasitization than other A. 7818R, exp 4. Discussion mellifera subspecies [53,54]. Again, the results are subjected to the above-mentioned limitations of the analysis including the n = 1 repetitions of the transcriptomic data per subspecies. We characterized VOV-1, a novel virus common to A. mellifera and V. destructor and VDV-4, a novel virus of V. destructor from A. cerana. VOV-1 possess a single-stranded negative-sense RNA genome and belongs to the Orthomyxoviridae family that among others includes the genus Thogotovirus. Most of the Thogotoviruses have been associated with ticks [55] and relatively few of them have been described in Acari or other types of hematophagous arthropods [56,57]. The VOV-1 genome has six segments and this is the ﬁrst report of the complete genome of a single-stranded negative-sense RNA segmented virus seen in honeybees and varroa mites. We provide evidence that VOV-1 replicates in individual varroa mites, and we found positive sense-virus RNA in pools collected from A. m. ligustica. Interestingly, it showed greater prevalence in V. destructor mites compared with honeybees of 78.57% and 8.33%, respectively, in samples from apiaries located at the North (Haifa, Kibbutz Lehavot HaBashan, Kibbutz Dan), the Center (ARO, Nitzanei Oz, Herut, Kfar Ruth), and the South (Kibbutz Viruses 2019, 11, 94 12 of 15 Yad Mordechai) of Israel. This is why, taking together the above data, we decided to name it Varroa orthomyxovirus-1, VOV-1. The discovery of these novel viruses in Apis mellifera and its recently acquired obligatory parasite Varroa destructor opens a new venue for investigation of viral interactions in honeybee colonies. A number of questions emerge concerning this new host–pathogen relationship that could interfere the preexisting balance. What is the pathology associated with VOV-1? Do varroa mites transmit VOV-1 and VDV-4 directly, e.g., transovarially, or via their host bee? Where do the viruses accumulate in varroa? Is VOV-1 infectious to A. mellifera? Furthermore, there is another issue concerning those viruses crucial for colony health and survival—DWV, ABPV, IAPV, and CBPV—and their interaction with newly discovered ARV1/ BRV-1, VOV-1, and VDV-2 and -3: Do they affect one another on a mutual base? If they do, on what level and what are the factors that may be involved (e.g., colony location, kind of treatment against varroa, season, colony resistance and/or hygienic behavior, etc.). 7818R, exp 4. Discussion Our ﬁndings and the tools that we have developed in this study pave the way to investigate these questions and extend our knowledge and understanding of the role played by viral pathogens in honeybee colonies. Supplementary Materials: The following are available online at http://www.mdpi.com/1999-4915/11/2/94/s1, Figure S1: title, Table S1: Table S1. Primers used for validation and quantitation of Varroa orthomyxovirus-1 (VOV-1). Table S2. Primers used for validation and quantitation of Varroa destructor virus-4 (VDV-4). Table S3. Complete VOV-1 nucleotide sequences. Table S4. Nucleotide sequence of VDV-4. Author Contributions: Conceptualization, S.L. and N.C.; Methodology, S.L., N.S., T.E., and D.N.; Software, N.S.; Validation, S.L and T.E.; Formal Analysis, S.L., N.S., D.N., and N.C.; Investigation, S.L., N.S., and N.C.; Resources, S.L., J.P., and T.E.; Data Curation, S.L. and N.S.; Writing—Original Draft Preparation, S.L. and P.N.; Writing—Review and Editing S.L., N.S., P.N., J.P., D.N., and N.C.; Visualization, S.L.,P.N., and N.C.; Supervision, N.C.; Project Administration and Funding Acquisition, N.C. Author Contributions: Conceptualization, S.L. and N.C.; Methodology, S.L., N.S., T.E., and D.N.; Software, N.S.; Validation, S.L and T.E.; Formal Analysis, S.L., N.S., D.N., and N.C.; Investigation, S.L., N.S., and N.C.; Resources, S.L., J.P., and T.E.; Data Curation, S.L. and N.S.; Writing—Original Draft Preparation, S.L. and P.N.; Writing—Review and Editing S.L., N.S., P.N., J.P., D.N., and N.C.; Visualization, S.L.,P.N., and N.C.; Supervision, N.C.; Project Administration and Funding Acquisition, N.C. Funding: The research was supported by a USAID Grant number TA-MOU-11-M32-035 and a Grant of the Chief Scientist of the Ministry of Agriculture of Israel number 131-1857. Acknowledgments: We thank Dr. Victoria Soroker, Mr. Yossef Kamer, and Mrs Nurit Eliash for their assistance with the honeybee colonies from ARO, and Rotem Zirler for her assistance with honeybee samples. Conﬂicts of Interest: The authors declare no conﬂicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. Conﬂicts of Interest: The authors declare no conﬂicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. References 1. Rosenkranz, P.; Aumeier, P.; Ziegelmann, B. Biology and control of Varroa destructor. J. Invertebr. Pathol. 2010, 103, S96–S119. [CrossRef] [PubMed] 2. Bowen-Walker, P.L.; Martin, S.J.; Gunn, A. The transmission of deformed wing virus between honeybees (Apis mellifera L.) by the ectoparasitic mite Varroa jacobsoni Oud. J. Invertebr. Pathol. 1999, 73, 101–106. [CrossRef] [PubMed] 3. Chen, Y.; Pettis, J.S.; Evans, J.D.; Kramer, M.; Feldlaufer, M.F. Transmission of Kashmir bee virus by the ectoparasitic mite Varroa destructor. 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[CrossRef] [PubMed] © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
https://openalex.org/W2562403933	https://europepmc.org/articles/pmc5334524?pdf=render	English	null	PAPP-A: a promising therapeutic target for healthy longevity	Aging Cell	2,016	cc-by	5,827	PAPP-A and lifespan studies Both male and female PAPP-A knockout (KO) mice on chow diet live 30–40% longer than wild-type (WT) littermates, with no secondary endocrine abnormalities (Conover et al., 2010b). Circulating levels of growth hormone (GH), IGF-I, glucose, and insulin were not signiﬁ- cantly different between PAPP-A KO and WT mice in this study. PAPP- A KO mice also live longer when fed a high fat diet starting as adults (Conover et al., 2015). Thus, PAPP-A deﬁciency can promote longevity without dietary restriction. Furthermore, this extended lifespan is not a secondary consequence of a small body size because PAPP-A KO mice rescued from the dwarf phenotype by enhanced IGF-II expression during fetal development retain their longevity advantage (Conover et al., 2010a). Finally, conditional knockout of the PAPP-A gene in adult mice using tamoxifen-inducible Cre recombinase methodology (Conover et al., 2013a) also resulted in a 20% extension of lifespan (P < 0.0001; manuscript in preparation). End-of-life pathology showed delayed occurrence of fatal neoplasias and indicated decreased incidence and severity of conditions with age-related degenerative changes, such as cardiomyopathy, nephropathy, and thymic atrophy in PAPP-A KO mice compared to WT littermates (Conover et al., 2010b). Further studies found PAPP-A to be expressed in many different cells types besides ﬁbroblasts, including human osteoblasts, vascular smooth muscle cells, pre-adipocytes, ovarian granulosa cells, and kidney mesangial cells (Lawrence et al., 1999; Conover et al., 2001, 2004b, 2006; Resch et al., 2004). It was characterized as a metalloproteinase of the metzincin superfamily and deﬁned a new subfamily, the pappalysins, distinct from previously recognized subfamilies including the matrix metalloproteinases (Boldt et al., 2001). Since the discovery of its function, PAPP-A and its proteolytic activity have been studied in a diverse range of species, including human, baboon, cow, pig, mouse, naked mole rat, marsupial, and zebraﬁsh (Mazerbourg et al., 2001; Conover et al., 2004a; Tchoukalova et al., 2009; Juengel et al., 2010; Phang et al., 2010; Kjaer-Sorensen et al., 2013; Brohus et al., 2015). Based on genomic data, a gene encoding PAPP-A appears to be present in all vertebrates. The main known function of PAPP-A is to increase local IGF bioavailability through cleavage of inhibitory IGFBPs, in particular IGFBP-4. Summary Pregnancy-associated plasma protein-A (PAPP-A) is a proteolytic enzyme that was discovered to increase local insulin-like growth factor (IGF) availability for receptor activation through cleavage of inhibitory IGF binding proteins (IGFBPs). Reduced IGF signaling has been associated with increased lifespan and healthspan. Therefore, inhibition of PAPP-A represents a novel approach to indirectly decrease the availability of bioactive IGF. Here, we will review data in support of PAPP-A as a therapeutic target to promote healthy longevity. Reduced IGF signaling has been associated with longevity and increased healthspan (Katic & Kahn, 2005). Therefore, a reduction in PAPP-A proteolytic activity represents a novel approach to indirectly decrease the availability of bioactive IGF. For therapeutic intervention, such a strategy is expected to moderately restrain IGF signaling and hence cause fewer adverse effects compared to direct inhibition by targeting the IGF receptor. Here, data in support for the development of PAPP-A as a therapeutic target are reviewed. These are principally based on studies with genetically modiﬁed mice and a recently developed monoclonal antibody speciﬁc for PAPP-A, which selectively inhibits PAPP- A cleavage of IGFBP-4 (Mikkelsen et al., 2008, 2014). The focus of this review is on curbing aging and age-related diseases. Key words: aging; IGF; PAPP-A. Introduction PAPP-A was ﬁrst identiﬁed as a placental protein of primates, but no biochemical function was known prior to our discovery in 1999 that PAPP-A is a proteolytic enzyme. We showed that cultured human ﬁbroblasts secrete PAPP-A, and that PAPP-A cleaves IGFBP-4 (Lawrence et al., 1999). Importantly, IGFBP-4 becomes a PAPP-A substrate only following its binding of IGF-I or -II. The two proteolytic fragments resulting from PAPP-A cleavage have very low afﬁnity for the IGFs, causing the IGFBP-4/IGF complex to dissociate. Doi: 10.1111/acel.12564 Doi: 10.1111/acel.12564 Aging Cell (2017) 16, pp205–209 REVIEW PAPP-A: a promising therapeutic target for healthy longevity Cheryl A. Conover1 and Claus Oxvig2 1Endocrinology, Mayo Clinic, Rochester, MN, USA 2Department of Molecular Biology and Genetics, Aarhus Universitet, Aarhus, Denmark 2008; Conover, 2012; Oxvig, 2015). Secreted PAPP-A tethers to cells through binding to surface glycosaminoglycans (Fig. 1). IGF bound to IGFBP-4 is not bioactive. However, the liberation of bioactive IGF upon cleavage of IGFBP-4 in the pericellular environment initiates IGF signaling. PAPP-A-induced enhancement of local IGF action through proteolysis of IGFBP-4 has been demonstrated in vitro and in vivo in several different systems (Conover, 2012; Oxvig, 2015). The only exception to date is in early fetal development of zebraﬁsh (Kjaer- Sorensen et al., 2013). Curiously, a paralog of IGFBP-4 appears to be lacking in zebraﬁsh (Li et al., 2009). ª 2016 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. Correspondence Cheryl A. Conover, Endocrinology, Mayo Clinic, 200 First Street SW5-194 Joseph, Rochester, MN 55905, USA. Tel: 507-255-6415; fax: 507-255-4828; e-mail: conover.cheryl@mayo.edu Accepted for publication 25 November 2016 PAPP-A and lifespan studies Indeed, PAPP-A is probably the only physiological IGFBP-4 proteinase (Conover et al., 2004a; Laursen et al., 2007; Ning et al., Aging Cell Several mouse models with reduced GH-stimulated IGF-I expression by liver and low levels of circulating IGF-I (Snell, Ames dwarf, GH receptor KO) have also been found to have extended longevity (Junnila et al., 2013). On the other hand, transgenic mice over-expressing GH exhibit a shortened lifespan (Bartke, 2003). It is important to note that PAPP-A KO mice have normal levels of circulating IGF-I (and GH) and their phenotype reﬂects reduction in local IGF action. Unlike the GH mutant mice that have postnatal growth retardation, deletion of the PAPP-A gene manifests itself early in fetal development as proportional Correspondence Cheryl A. Conover, Endocrinology, Mayo Clinic, 200 First Street SW5-194 Joseph, Rochester, MN 55905, USA. Tel: 507-255-6415; fax: 507-255-4828; e-mail: conover.cheryl@mayo.edu Accepted for publication 25 November 2016 205 ª 2016 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. PAPP-A & Healthy Longevity, C. A. Conover and C. Oxvig 206 206 Fig. 1 Role of PAPP-A and IGFBP-4 in the local control of IGF signaling. deletion in ApoE KO mice inhibited atherosclerotic plaque burden by 70– 80% following 20 weeks on a high fat diet. There was no effect on macrophage recruitment or expression of pro-inﬂammatory cytokines, and no secondary endocrine effects were observed (Harrington et al., 2007). Tang et al. (2012) suggested that PAPP-A inhibits cholesterol efﬂux in macrophage-derived foam cells through the IGF-I signaling pathway, thus contributing to the pathogenesis of atherosclerosis. To assess a possible effect of PAPP-A gene knockout during development, a similar experiment was carried out in ApoE KO mice with the PAPP-A gene ‘ﬂoxed’ and crossed with mice with tamoxifen- inducible Cre recombinase (Conover et al., 2013a). Inducible reduction in PAPP-A gene expression in adult ApoE KO mice signiﬁcantly inhibited established atherosclerotic plaque progression and the development of advanced plaque with necrotic cores (Bale et al., 2014). In this inducible model, reduced PAPP-A expression was not limited to the vasculature, and changes in other tissues could potentially contribute to the observed effects. PAPP-A and lifespan studies PAPP-A gene expression was efﬁciently reduced in thymus and spleen (Conover et al., 2013a), which could dampen the negative effects of the immune response in atherosclerosis (Hansson & Libby, 2006). Moreover, weekly treatment (intraperitoneal injections) of ApoE KO mice with mAb-PA, which speciﬁcally inhibits PAPP-A-mediated IGFBP-4 proteolysis, similarly inhibited atherosclerotic plaque progression (Conover et al., 2016b). This latter study was the ﬁrst to demonstrate proof of principle and provide translational support for the development of novel therapeutic strategies to inhibit human atherosclerosis by targeting the proteolytic activity of PAPP-A. Fig. 1 Role of PAPP-A and IGFBP-4 in the local control of IGF signaling. dwarﬁsm (Conover et al., 2004a). The lifespan extension in the Snell, Ames dwarf, and GH receptor KO models reﬂects GH tone rather than IGF-I bioavailability. Thymic involution Thymic involution, the shrinking and morphological changes of the thymus in aging vertebrates, results in immune senescence that has the potential to impact aging and age-related diseases. As reported previously, the thymi of PAPP-A KO mice are relatively resistant to normal age-dependent involution (Vallejo et al., 2009). At 18 months of age, thymi of WT mice are markedly reduced in size and cellularity with extensive adipose inﬁltration. In contrast, the similarly aged PAPP-A KO mice maintain the lobular thymic structure and cellularity and the ability to produce diverse immune-competent T cells. We recently completed a study of young PAPP-A KO and WT mice that were fed a high fat diet for 20 weeks. Compared to WT littermates, the thymi of male and female PAPP-A KO mice were 122% (P = 0.007) and 158% (P < 0.0001) larger, respectively. Even with the larger size, the thymi of female PAPP-A KO mice contained 40% less lipid (P = 0.033) than WT mice. In a preliminary study, once a week intraperitoneal treatment of WT mice on high fat diet with an inhibitory monoclonal antibody against PAPP-A (mAb-PA) resulted in thymic size that was 120% of treatment with isotype control antibody. Targeting PAPP-A to reduce/delay thymic involution could, therefore, play a central role in regulating immune competence and healthy aging. However, further studies are needed to better characterize and understand the role of PAPP-A in thymic biology. Visceral obesity In contrast to subcutaneous fat, the accumulation of visceral fat is strongly associated with severe metabolic complications in humans (Wajchenberg, 2000; Jensen, 2008; Perrini et al., 2008), which shows differential expression of PAPP-A in these two types of adipose tissue (Tchoukalova et al., 2009; Davidge-Pitts et al., 2014). Mouse adipose tissue shows a similar difference with ﬁvefold to 10-fold higher expression of PAPP-A mRNA in visceral (mesenteric) fat compared to subcutaneous (inguinal, subscapular) fat. There was a preferential impact of PAPP-A deﬁciency in mice on a high fat diet to prevent increases in mesenteric adipocyte size with no signiﬁcant effect on subcutaneous fat depots, probably due to the high expression of PAPP-A in mesenteric fat (Conover et al., 2013b). The decrease in accumulation PAPP-A and age-related diseases It is of note that PAPP-A can also cleave IGFBP-5 (Laursen et al., 2007). However, as there are other proteases that have IGFBP-5 as substrate, the impact of PAPP-A-speciﬁc IGFBP-5 proteolysis is difﬁcult to ascertain in vivo. The inhibitory mAb-PA was selected to target a speciﬁc region of PAPP-A required for proteolytic cleavage of IGFBP-4, but not IGFBP-5 (Boldt et al., 2004; Mikkelsen et al., 2008, 2014), a so-called substrate-binding exosite. Therefore, mAb-PA inhibits proteolysis of IGFBP-4, but not IGFBP-5. This immunoneutralizing antibody was effective in inhibiting atherosclerotic plaque progression (Conover et al., 2016b). In addition, ApoE KO mice expressing a mutated PAPP-A, which selectively lacks proteolytic activity against IGFBP-4 and not IGFBP-5, did not show accelerated atherosclerotic lesion development compared to mice overexpressing native PAPP-A (Boldt et al., 2013). Interestingly, neither did ApoE KO mice expressing proteolytically active PAPP-A, but lacking domains for cell association. Thus, data so far support the model set forth by Laursen et al. (2007) that although cleavage of both IGFBP-4 and IGFBP-5 is required for release of bioactive IGF, cell surface-localized proteolysis of IGFBP-4 represents the ﬁnal regulatory step of efﬁcient IGF delivery to receptors. Fragility Aging is associated with loss of skeletal muscle mass and, consequently, compromised muscle function (Marzetti et al., 2009). It has been found that PAPP-A KO mice are resistant to skeletal muscle aging (Conover et al., 2016a). In this study, top genes regulated in muscle from 18-month-old PAPP-A KO compared to WT mice were associated with increased muscle function, increased lipid metabolism, and decreased stress. Fiber cross-sectional area and intrinsic mitochondrial oxidative capacity were signiﬁcantly increased in skeletal muscle of aged PAPP-A KO compared to WT mice. Moreover, these 18-month-old PAPP-A KO mice exhibited signiﬁcantly enhanced endurance running on a treadmill. Also, PAPP-A KO mice are resistant to age-related metabolic dysfunction (Hill et al., 2015), largely driven by skeletal muscle. These studies may seem contrary to other studies where PAPP-A overexpression in skeletal muscle increased muscle mass in response to Injury (Rehage et al., 2007). It is known that enhanced IGF signaling is important for muscle regeneration in response to injury (Clemmons, 2009). However, this response could be different in aging muscle. One possibility is that reduced IGF signaling in pluripotent stem cells residing in skeletal muscle protects against premature depletion. More work is required to delineate the roles of IGFs in this tissue. Atherosclerosis As ﬁrst reported by Bayes-Genis et al. (2001), PAPP-A expression is increased in unstable human atherosclerotic plaques associated with activated macrophages, smooth muscle cells, and endothelium. To assess whether this increased PAPP-A expression was cause or conse- quence of lesion development, we studied apolipoprotein E (ApoE) KO mice, which are an established murine model of atherosclerosis (Breslow, 1996). Reduction in local IGF bioavailability through PAPP-A gene ª 2016 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd. PAPP-A & Healthy Longevity, C. A. Conover and C. Oxvig 207 shown to have increased tumor aggressiveness in vivo (Boldt & Conover, 2011). The ﬁrst report of mAb-PA efﬁcacy was in lung cancer cell xenografts expressing PAPP-A (Mikkelsen et al., 2014). PAPP-A is also overexpressed in a subset of human ovarian tumors, and weekly treatment of mice harboring these intraperitoneal patient tumorgrafts with the inhibitory antibody, mAb-PA, could reduce tumor size and ascites burden. Treatment with mAb-PA also conferred chemo-sensitivity to a high PAPP-A-expressing chemo-resistant patient tumor (Becker et al., 2015). These beneﬁcial effects of inhibiting the proteolytic activity of PAPP-A were only manifested in tumors expressing moderate-to-high levels of PAPP-A. Thus, PAPP-A expression in patient tumors, or ascites (Thomsen et al., 2015), may be a biomarker for patient response to PAPP-A therapies. We also have unpublished data indicating that mAb- PA treatment can prevent ovarian tumor metastases to intraperitoneal lining and perigonadal fat, these being prime sites for metastases via ascites. This ﬁts with the identiﬁcation of PAPP-A as a migration/ invasion-promoting gene in an ovarian cancer cell line (Boldt & Conover, 2011), malignant pleural mesothelioma cells (Huang et al. 2013), nonsmall cell lung cancer (Salim et al., 2013), and melanoma (Prithviraj et al. 2015). of mesenteric adipose tissue was associated with increased expression of adiponectin, which has anti-inﬂammatory and cardio-protective effects and enhanced insulin receptor signaling in this depot (Conover et al., 2013b). For these reasons, PAPP-A may be a potential target for treatment and/or prevention strategies for visceral obesity and related morbidities. Inﬂammation Aging and many age-related diseases are associated with inﬂammation. However, the use of anti-inﬂammatory drugs to alleviate these condi- tions is problematic because they affect multiple pathways and lack speciﬁcity. For example, cyclooxygenase-2 inhibition was used to counteract inﬂammation, but was associated with serious cardiovascular complications (Sohn & Krotz, 2006). Furthermore, inﬂammation is important in normal tissue repair and defense against infection. Thus, it is generally recommended that targets should be speciﬁc and down- stream of inﬂammatory pathways (Gauldie, 2007). PAPP-A could be such a target. Cancer The involvement of PAPP-A proteolytic activity in cancer is increasingly suggested. For example, clinical data show that PAPP-A serum levels are elevated in patients with lung cancer (Bulut et al., 2009), and PAPP-A can promote lung cancer growth in vivo (Pan et al. 2012). PAPP-A is highly expressed in aggressive forms of human breast cancer (Mansﬁeld et al., 2014). Interestingly, Chander et al. (2011) found that mutation of the tumor suppressor gene, p53, increases PAPP-A transcription in breast cancer cell lines and mammary gland tissues. Nephropathy The kidney has been suggested to be an organ of particular interest for targeted PAPP-A inhibition (Swindell et al., 2010). At 18 months of age, there was a higher incidence and severity of chronic nephropathy in WT compared to PAPP-A KO mice (Conover et al., 2010b). It was also found that PAPP-A KO mice are resistant to the development of diabetic nephropathy (Mader et al., 2013). In this model of type 2 diabetes, increased glomerular size and thickened Bowman’s capsules were observed in WT mice, but not in PAPP-A KO mice. Glomerular expression of PAPP-A was increased in kidneys of patients with diabetic nephropa- thy (Mader et al., 2013). Studies in cultured human mesangial cells indicated potent upregulation of PAPP-A mRNA and protein expression by pro-inﬂammatory cytokines (Donegan et al., 2016), which are prevalent in diabetic nephropathy. The pro-inﬂammatory cytokines, interleukin-1b (IL-1b), and tumor necrosis factor-a (TNF-a) are potent stimulators of PAPP-A expression in human ﬁbroblasts, osteoblasts, arterial smooth muscle cells, endothelial cells, pre-adipocytes, intervertebral disk cells, and mesangial cells (Conover et al., 2004b, 2006, 2008; Resch et al., 2004; Gruber et al., 2013; Davidge-Pitts et al., 2014). In atherosclerotic plaque, macro- phage-derived pro-inﬂammatory cytokines act in a paracrine manner to stimulate vascular smooth muscle and endothelial cells to secrete PAPP-A (Conover et al., 2007). This represents an important ampliﬁcation point in plaque progression, as PAPP-A can feedback back on activated macrophages and smooth muscle cells to promote IGF-mediated lipid uptake. With the loss of the PAPP-A activity induced by pro- inﬂammatory cytokines, the vicious cycle would be blunted in spite of the continued presence of macrophages and similar levels of IL-1b and TNF-a expression. We suggest that this model of PAPP-A and inﬂam- matory stress may also apply to other conditions. Aging, associated with chronic low-grade inﬂammation, is one such condition. With visceral fat representing a highly active inﬂammatory microenvironment, especially in obese subjects, adipose tissue dysregulation is another. ª 2016 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd. Contraindications for PAPP-A-directed therapies Low circulating PAPP-A has been associated with adverse effects on placental function and fetal growth in humans (Smith et al., 2002). Although the role of PAPP-A in human pregnancy is not understood, PAPP-A is believed to be important for placental development. There- fore, targeting PAPP-A during human pregnancy is not likely to be a In agreement with such ﬁndings, several publications indicate that targeting of PAPP-A proteolytic activity is relevant to the prevention of cancer growth and metastases. Overexpression of proteolytically active, but not proteolytically inactive, PAPP-A in an ovarian cancer cell line was ª 2016 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd. PAPP-A & Healthy Longevity, C. A. Conover and C. Oxvig 208 viable strategy. Interestingly, placental development in mice does not depend on PAPP-A (Qin et al., 2002; Soe et al., 2002). Boldt HB, Conover CA (2011) Overexpression of pregnancy-associated plasma protein-A in ovarian cancer cells promotes tumor growth in vivo. Endocrinology 152, 1470–1478. The involvement of PAPP-A in normal tissue repair processes also suggests a possible need to suspend PAPP-A targeting temporarily during such conditions. For example, PAPP-A increases bone accretion primarily by increasing IGF bioavailability important for prepubertal bone growth (Mohan et al., 2003; Qin et al., 2006). Fracture repair in PAPP-A KO mice is temporally compromised, but not prevented from normal resolution (Miller et al., 2007). Similarly, controlled increases in PAPP-A expression are seen in healing human skin (Chen et al., 2003), indicating that wound healing may be delayed as a consequence of PAPP-A targeting. Boldt HB, Overgaard MT, Laursen LS, Weyer K, Sottrup-Jensen L, Oxvig C (2001) Mutational analysis of the proteolytic domain of pregnancy-associated plasma protein-A (PAPP-A): classiﬁcation as a metzincin. Biochem. J. 358, 359–367. Boldt HB, Kjer-Sorensen K, Overgaard MT, Weyer K, Poulsen CB, Sottrup-Jensen L, Conover CA, Giudice LC, Oxvig C (2004) The Lin12-notch repeats of preganancy-associated plasma protein-A bind calcium and determine its proteolytic speciﬁcity. J. Biol. Chem. 279, 38525–38531. Boldt HB, Bale LK, Resch ZT, Oxvig C, Overgaard MT, Conover CA (2013) Effects of mutated pregnancy-associated plasma protein-a on atherosclerotic lesion development in mice. Endocrinology 154, 246–252. Breslow JL (1996) Mouse models of atherosclerosis. Science 272, 685–688. Brohus M, Gorbunova V, Faulkes CG, Overgaard MT, Conover CA (2015) The insulin-like growth factor system in the long-lived naked mole-rat. PLoS ONE 10, e0145587. Conclusion Conover CA, Chen B-K, Resch ZT (2004b) Regulation of pregnancy-associated plasma protein-A expression n cultured human osteoblasts. Bone 34, 297–302. Author contributions Conover CA, Bale LK, Harrington SC, Resch ZT, Overgaard MT, Oxvig C (2006) Cytokine stimulation of pregnancy-associated plasma protein A expression in human coronary artery smooth muscle cells: inhibition by resveratrol. Am. J. Physiol. Cell Physiol. 290, C183–C188. Drs. Conover and Oxvig contributed equally to the preparation of this review. Conover CA, Harrington SC, Bale LK, Oxvig C (2007) Surface association of pregnancy-associated plasma protein-A accounts for its colocalization with activated macrophages. Am. J. Physiol. Heart Circ. Physiol. 292, H994–H1000. Conclusion Bulut I, Coskun A, Ciftci A, Cetinkaya E, Altiay G, Caglar T, Gulcan E (2009) Relationship between pregnancy-associated plasma protein-A and lung cancer. Am. J. Med. Sci. 337, 241–244. Experimental evidence is accumulating that inhibition of PAPP-A has the potential to promote healthy longevity. It is clearly advantageous that targeting of PAPP-A has the beneﬁt of a single intervention that affects multiple adverse changes with age, not just a single condition (Figueira et al., 2016). PAPP-A is present in the extracellular environment, and its activity is therefore amenable to pharmacologic intervention. Strategies to inhibit PAPP-A have recently been developed and tested in experi- mental models (Mikkelsen et al., 2008, 2014; Becker et al., 2015; Conover et al., 2016b). Rather than the active site of PAPP-A, a unique substrate-binding exosite, critical for proteolytic cleavage of IGFBP-4, is targeted. This efﬁciently eliminates activity toward IGFBP-4, but does not interfere with cleavage of other possible substrates of PAPP-A. Inhibition will target discrete conditions with increased PAPP-A activity, resulting in moderate restraint of IGF signaling and minimizing side effects. However, much remains to be learned about stages in life at which mice, and possibly humans, are susceptible to improvements in long- term health by manipulation of PAPP-A. Chander H, Halpern M, Resnick-Silverman L, Manfredi JJ, Germain D (2011) Skp2B overexpression alters a prohibitin-p53 axis and the transcription of PAPP-A, the protease of insulin-like growth factor binding protein 4. PLoS ONE 6, e22456. Chen B-K, Leiferman KM, Pittelkow MR, Overgaard MT, Oxvig C, Conover CA (2003) Localization and regulation of pregnancy associated plasma protein-A expression in healing human skin. J. Clin. Endocrinol. Metab. 88, 4465–4471. Clemmons DR (2009) Role of IGF-I in skeletal muscle mass maintenance. Trends Endocrinol. Metab. 20, 349–356. Conover CA (2012) Key questions and answers about pregnancy-associated plasma protein-A. Trends Endocrinol. Metab. 23, 242–249. Conover CA, Faessen GF, Ilg K, Chandrasekher YA, Christiansen M, Overgaard MT, Oxvig C, Giudice LC (2001) Pregnancy-associated plasma protein-A is the insulin- like growth factor binding protein-4 protease secreted by human ovarian granulosa cells and is a marker of dominant follicle selection and the corpus luteum. Endocrinology 142, 2155–2158. Conover CA, Bale LK, Overgaard MT, Johnstone EW, Laursen UH, Fuchtbauer E-M, Oxvig C, van Deursen J (2004a) Metalloproteinase pregnancy-associated plasma protein A is a critical growth regulatory factor during fetal development. Development 131, 1187–1194. Conﬂict of interest Conover CA, Bale LK, Mader JR, Mason MA, Keenan KP, Marler RJ (2010b) Longevity and age-related pathology of mice deﬁcient in pregnancy-associated Conover CA, Bale LK, Mader JR, Mason MA, Keenan KP, Marler RJ (2010b) Longevity and age-related pathology of mice deﬁcient in pregnancy-associated plasma protein-A. J. Gerontol. A Biol. Sci. Med. Sci. 65, 590–599. None declared. Conover CA, Bale LK, Powell DR (2013a) Inducible knock out of pregnancy- associated plasma protein-A gene expression in the adult mouse: effect on vascular injury response. Endocrinology 154, 2734–2738. Funding This work was supported by NIA grant AG028141 (CAC) and The Novo Nordisk Foundation (CO). Conover CA, Harrington SC, Bale LK (2008) Differential regulation of pregnancy associated plasma protein-A in human coronary artery endothelial cells and smooth muscle cells. Growth Horm. IGF Res. 18, 213–220. Conover CA, Bale LK, Grell JA, Mader JR, Mason MA (2010a) Longevity is not inﬂuenced by prenatal programming of body size. Aging Cell 9, 647–649. ª 2016 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd. References Cancer 52, 895–911. 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https://openalex.org/W4309449299	https://dergipark.org.tr/tr/download/article-file/2100073	Turkish	null	Determination of Master's Students' Views in Science Education on STEM Education	DergiPark (Istanbul University)	2,021	cc-by	7,328	Received: 11.25.2021 Received: 11.25.2021 Received: 11.25.2021 Accepted: 12.10.2021 Article Type: Ethics Declaration: EURASIAN JOURNAL OF TEACHER EDUCATION Year: 2021 Volume: 2 Number: 3 Pages: 221-237 Determination of Master’s Students’ Views in Science Education on STEM Education Fatih Gürbüz1, Işın Kahveci2 1 Bayburt University, Türkiye, fgurbuz@bayburt.edu.tr, ORCID: 0000-0001-9200-9202 2 Bayburt University, Türkiye, kahvecisin@hotmail.com, ORCID: 0000-0001-7974-5723 To cite this article: Gürbüz, F. & Kahveci, I. (2021). Determination of Master's Students’ Views in Science Education on STEM Education. Eurasian Journal of Teacher Education, 2(3), 221-237. EURASIAN JOURNAL OF TEACHER EDUCATION Year: 2021 Volume: 2 Number: 3 Pages: 221-237 Determination of Master’s Students’ Views in Science Education on STEM Education Fatih Gürbüz1, Işın Kahveci2 1 Bayburt University, Türkiye, fgurbuz@bayburt.edu.tr, ORCID: 0000-0001-9200-9202 2 Bayburt University, Türkiye, kahvecisin@hotmail.com, ORCID: 0000-0001-7974-5723 To cite this article: Gürbüz, F. & Kahveci, I. (2021). Determination of Master's Students’ Views in Science Education on STEM Education. Eurasian Journal of Teacher Education, 2(3), 221-237. Pages: 221-237 To cite this article: Gürbüz, F. & Kahveci, I. (2021). Determination of Master's Students’ Views in Science Education on STEM Education. Eurasian Journal of Teacher Education, 2(3), 221-237. Abstract The aim of this study is to determination of master's students’ views in science education on STEM education. For this purpose, the sample of the study consists of 18 students who are in the course of taking a course and are doing their master's degree in the Mathematics and Science Education Department / Science Education Master's Program in the fall semester of the 2021-2022 academic year. 11 of the participants are female and 7 of them are male students. The case study method, one of the qualitative research methods, was used in the study. The data obtained within the scope of the study were taken with a semi-structured interview form created by an expert and researcher. The obtained data were analyzed by content analysis method. As a result of the research, it was concluded that STEM education had a positive effect on students, increased meaningful learning, and improved problem solving and high-level thinking skills. On the other hand, it was determined that the participants did not have sufficient content knowledge in STEM education. Based on the results of the research, it has been suggested that education faculties should innovate in their curricula in order to provide adequate STEM education. Keywords: Science education, STEM education, Master students etermination of Master’s Students’ Views in Science Education on STEM Education 1 Bayburt University, Türkiye, fgurbuz@bayburt.edu.tr, ORCID: 0000-0001-9200-9202 2 Bayburt University, Türkiye, kahvecisin@hotmail.com, ORCID: 0000-0001-7974-5723 Ethics Declaration: The author declared that he complies with all ethical rules. Bayburt University Ethics Committee has approved the protocol of this study approved of ethical approval dated 11.08.2021 and number of 170. Fen Bilgisi Eğitimi Yüksek Lisans Öğrencilerinin STEM Eğitimi Hakkındaki Görüşlerinin Belirlenmesi Öz Bu çalışmanın amacı, Fen Bilgisi Eğitimi yüksek lisans öğrencilerinin STEM eğitimi hakkındaki görüşlerinin belirlenmesidir. Bu amaç doğrultusunda çalışmanın örneklemini 2021-2022 Eğitim- Öğretim yılı güz döneminde Matematik ve Fen Bilimleri Eğitimi Anabilim Dalı / Fen Bilgisi Eğitimi Tezli Yüksek Lisans Programında yüksek lisans yapan ve ders alma döneminde olan 18 öğrenci oluşturmaktadır. Katılımcıların 11 tanesi kadın, 7 tanesi ise erkek öğrencilerden oluşmaktadır. Çalışmada nitel araştırma yöntemlerinden durum çalışması yöntemi kullanılmıştır. Çalışma kapsamında elde edilen veriler bir uzman ve araştırmacı tarafından oluşturulan yarı yapılandırılmış görüşme formuyla alınmıştır. Elde edilen veriler içerik analizi yöntemiyle çözümlenmiştir. Araştırma sonucunda STEM eğitiminin öğrenciler üzerinde olumlu etki bıraktığı, anlamlı öğrenmeyi arttırdığı, problem çözme ve üst düzey düşünme becerisini geliştirdiği sonucuna ulaşılmıştır. Diğer taraftan, katılımcıların STEM eğitiminde yeterli alan bilgisine sahip olmadıkları tespit edilmiştir. Araştırma sonuçlarından yola çıkarak eğitim fakültelerinin yeterli seviyede STEM eğitimi verebilmesi için müfredatlarında yeniliğe gitmeleri önerilmiştir. Anahtar Kelimeler: Fen bilgisi eğitimi, STEM eğitimi, Yüksek lisans öğrencileri. Giriş Gelişen teknolojiyle birlikte hayatımızda birçok değişiklik olmuştur. Günlük, sıradan aktiviteleri gerçekleştirirken bile teknolojiden yardım almaktayız. Navigasyon, güvenli veri iletimi, hızlı iletişim bunlardan sadece birkaçı. Toplantılarımız, yapacağımız bir sonraki iş hamlemiz ve planlarımız bile artık, teknoloji sayesinde, anında bize hatırlatılıp hızlı bir şekilde hayat buluyor. Teknolojinin hayatımıza bu denli girmesiyle; analitik, eleştirel ve yenilikçi düşünebilen, problem çözme becerisi yüksek, tasarlayan, bilgiye ulaşırken teknolojiyi nasıl kullanması gerektiğini bilen, öz yeterlik bilinci yüksek, üreten, sorgulayan ve teknoloji okuryazarı olan bireylere gereksinim ihtiyacı artmaktadır (Kaya, Şahin & Uz-Özkılıç, 2020). Bu özelliklere sahip nitelikli bireyler yetiştirmek için mühendislik becerileri ve teknoloji ortaokullarda uygulanmaya başlanmış, STEM olarak adlandırılan eğitim hızlı bir şekilde yaygınlaştırılmıştır (Yıldırım, 2018). Yıldırım ve Altun’a (2015) göre STEM; öğrenilen bilgiyi günlük yaşamla entegre ederek anlamlı öğrenmeyi destekleyen ve 21. yüzyıl becerilerinin gelişimine olumlu katkı sağlayan bir yaklaşımdır (Yıldırım & Türk, 2018). STEM kelimesi “Science”, “Technology”, “Engineering” ve “Mathematics” kelimelerinin baş harflerinin bir araya getirilmesiyle oluşturulan bir ifadedir (Gonzalez & Kuenzi, 2012). STEM, okul öncesi eğitimden üniversiteye kadar uzanan ve farklı disiplinleri birleştiren bir yaklaşım olarak kabul görmektedir (Gonzalez & Kuenzi, 2012). Öğrenciler okul öncesinden başlayarak STEM eğitimi alırlarsa daha en başından problemlerini kolayca çözebilen, iyi iletişim kurabilen ve bir olayı bütün yönleriyle düşünebilen bireyler olarak yetişebilirler (Kaya, Şahin & Uz-Özkılıç, 2020). STEM eğitiminin etkili ve doğru bir şekilde uygulanması, öğrencilerin ilgi duyup başarılı olmaları için öğretmenlerin yeterli düzeyde alan bilgisi, beceri ve donanıma sahip olması gerekmektedir (Yıldırım & Türk, 2018). STEM eğitimi bakımından donanımlı öğretmen yetiştirmek; fen konularının yanında mühendislik ve teknolojiyi içine alan bir öğretim programı kullanımıyla sağlanabilir (Bakırcı & Kutlu, 2018). Bu bağlamda 2018’de yenilenen Fen Bilimleri Öğretim Programı’na göre STEM uygulamaları, öğrencilerin mühendislik ve bilim arasındaki bağlantıyı kurmalarına, disiplinler arası etkileşimi anlamalarına ve öğrendiklerini yaşama entegre etmelerine katkı sağlamaktadır (Milli Eğitim Bakanlığı [MEB], 2018). Fen Bilimleri Öğretim Programı’nın amacına uygun şekilde öğrencilerin değişen ve sürekli gelişen eğitimle beraber girişimci ve inovatif düşünebilen bireylere dönüştüğü görülmektedir (Bakırcı & Kutlu, 2018). STEM eğitiminde inovasyonun gereksinimlerine karşılık verebilmek adına, ülkemizde ve dünyada yaşanan teknolojik gelişmeleri 223 GÜRBÜZ ET AL. takip etme ve sorgulama becerisine sahip olma yetkinlikleri öne çıkar (Türk Sanayicileri ve İş İnsanları Derneği [TÜSİAD], 2017). Öğrencilere kazandırılmak istenen bu beceriler fen ve mühendislik uygulamalarının deneyimlenmesiyle oluşabilmektedir (MEB, 2018). Yapılan çalışmalarda, çeşitli seviyelerde öğrenim görmekte olan öğrencilerin fen bilimleri dersinde STEM uygulamalarının artmasıyla birlikte akademik başarılarının da arttığı gözlemlenmiştir (Ergün & Balçın, 2018; Güven, Selvi & Benzer, 2018). Giriş Son güncel veriler incelendiğinde matematik ve fen derslerinde ortalama başarıda anlamlı ölçüde artmalar meydana gelmiştir. Fen değerlendirmesinde ilk kez ölçek orta noktasının (500 puan) üzerinde başarı sağlanmıştır (MEB, 2020; Trends in İnternational Mathematics and Science Study [TİMSS], 2019). STEM eğitiminin amacına uygun şekilde yapılmasında öğretmenler kritik bir öneme sahiptir. Bundan dolayı öğretmenlerin STEM eğitimi ile ilgili düşüncelerinin ortaya çıkarılması ve sınıf ortamında uygulanabilirliğinin belirlenmesi gerekmektedir (Eroğlu & Bektaş, 2016). Araştırmanın öğretmenlerin bu alandaki eksikliklerini giderebileceği ve fen bilimleri eğitimi üzerine yüksek lisansa başlayacak öğretmenlere yardımcı olacağı düşünülmektedir (Eroğlu & Bektaş, 2016). Alanyazın incelendiğinde öğretmenlerin STEM eğitimine yönelik görüşlerinin alınması üzerine birçok çalışma mevcuttur (Bakırcı & Kutlu, 2018; Güldemir & Çınar, 2017; Kaya, Şahin & Uz-Özkılıç, 2020; Sözen & Alagöz, 2021). Ancak fen bilgisi eğitimi yüksek lisans öğrencilerinin STEM eğitimi hakkındaki görüşlerinin belirlendiği çalışmalar daha çok mevcut çalışmaların incelenmesi şeklindedir (Ceylan, 2021; Elmalı & Balkan-Kıyıcı, 2017; Herdem & Ünal, 2018). Oysaki yeterli düzeyde STEM eğitimi almış veya sınıfında uygulamış fen bilimleri yüksek lisans öğrencileri bu konuyla alakalı alanyazına olumlu katkı sağlayabilir. Bundan dolayı araştırmanın amacı, fen bilimleri öğretmenliği yüksek lisans öğrencilerinin STEM eğitimi hakkındaki görüşlerinin belirlenmesidir. Bu amaç doğrultusunda “Fen bilimleri öğretmenliği yüksek lisans öğrencilerinin STEM eğitimi hakkındaki görüşleri nelerdir?” problem durumuna cevap aranmıştır. Araştırma Deseni Araştırmada fen bilgisi eğitimi yüksek lisans öğrencilerinin STEM eğitimi hakkındaki görüşlerinin belirlenmesi amaçlanmaktadır. Bu amaç doğrultusunda çalışmada nitel araştırma yöntemlerinden durum çalışması yöntemi kullanılmıştır. Durum çalışması, araştırmacının belli bir zaman dilimi içerisinde sınırlandırılmış durumları derinlemesine incelendiği, durumların ve duruma bağlı temaların tanımlandığı nitel bir araştırma yaklaşımıdır (Creswell, 2003). Durum çalışmasının en belirgin özelliği ise, konu üzerinde ayrıntılı inceleme yapılmasına olanak sağlamasıdır. Yani işlenen konuyu bütüncül yaklaşımla gözler önüne koyar. Araştırmada incelenen durum fen bilimleri öğretmenliği yüksek lisans öğrencilerinin STEM eğitimi hakkındaki görüşleridir. Araştırma Grubu Araştırmanın çalışma grubunu 2021-2022 eğitim-öğretim yılı güz döneminde Bayburt Üniversitesi Lisansüstü Eğitim Enstitüsü’nde ders alma döneminde olan 18 fen bilgisi eğitimi yüksek lisans öğrencisi oluşturmaktadır. Katılımcılar 11 kadın ve 7 erkekten oluşmaktır. Kadın katılımcıların 9 tanesi, erkek katılımcıların ise 6 tanesi çeşitli kurumlarda fen bilimleri öğretmenliği yapmaktadır. Katılımcılar belirlenirken seçkisiz olmayan örnekleme yöntemlerinden amaçlı örnekleme yöntemi kullanılmıştır. Buna benzer çalışma gruplarında zaman, yer ve konum gibi özelliklerin yanında konunun uygulanabilirliği de önemlidir (Yıldırım & Selvi, 2018). Sorular 1) STEM hangi disiplinlerle ilişkilidir? 2) STEM denildiğinde aklınıza neler gelmektedir? 2) STEM denildiğinde aklınıza neler gelmektedir? 3) STEM eğitiminin fen öğretimine katkısı olduğunu düşünüyor musunuz, kısaca açıklay 3) STEM eğitiminin fen öğretimine katkısı olduğunu düşünüyor musunuz, kısaca açıklayınız? 4) Yüksek lisans eğitimi sırasında alanyazında STEM ile ilgili yapılmış güncel yayınları takip 3) STEM eğitiminin fen öğretimine katkısı olduğunu düşünüyor musunuz, kısaca açıklayınız? 4) Yüksek lisans eğitimi sırasında alanyazında STEM ile ilgili yapılmış güncel yayınları takip ediyor musunuz? 4) Yüksek lisans eğitimi sırasında alanyazında STEM ile ilgili yapılmış güncel yayınları takip ediyor musunuz? ) Alanyazını incelediğinizde STEM ile ilgili yapılan çalışmaları yeterli buluyor musunuz? 6) Yüksek lisans eğitimi süresince STEM eğitimine yönelik ders aldınız mı? STEM bilginizin yeterli olduğunu düşünüyor musunuz? 7) Yüksek lisans tez konunuzun STEM eğitimi üzerine olmasını tercih eder misiniz? 8) Öğretmenlik uygulaması dersi kapsamındaki uygulamalarınızda veya öğretmenlik mesleğinizde STEM etkinliklerini kullanarak neler yaptınız? STEM etkinliklerini kullanmadıysanız neden kullanmadınız? Fen bilimleri dersinde STEM etkinlikleri yaptırmanın avantaj ve dezavantajları nelerdir? 9) Fen bilimleri dersinde STEM etkinlikleri yaptırmanın avantaj ve dezavantajları neler 10) Fen bilimleri dersinde STEM etkinliklerini arttırmak için hangi çalışmalar yapılmalıdır? 0) Fen bilimleri dersinde STEM etkinliklerini arttırmak için hangi çalışmalar yapılmalıdır? VIEWS IN SCIENCE EDUCATION ON STEM EDUCATION 224 Verilerin Toplanması Araştırmada veri toplama aracı olarak, araştırmacı ve bir uzman tarafından oluşturulan yarı yapılandırılmış görüşme formu kullanılmıştır. Sorular hazırlanırken alanyazın taraması yapılmıştır. Oluşturulan sorular uzman görüşleri alınarak düzenlenmiş ve bazı sorularda değişiklikler yapılmıştır. Gerekli düzenlemeler yapıldıktan sonra 10 sorudan oluşan görüşme formuna son şekli verilmiştir. Araştırmacı ve uzman tarafından oluşturulan görüşme formu öğrencilere yöneltilerek cevapları yazmaları istenmiştir. Elde edilen verilerin analizinde ve değerlendirilmesinde farklı araştırmacıların da görüşleri alınmıştır. Araştırmanın güvenilirliğini arttırmak için katılımcılar, çalışmaya katılmak isteyen, gönüllü, yüksek lisans öğrencilerinden seçilmiştir. Araştırmanın geçerliğini sağlamak için, katılımcıların onayı alınmış aynı zamanda araştırmacı üçgenlemesi yapılmıştır (Bakırcı & Kutlu, 2018). Çalışmanın iç geçerliğini artırmak için ise çalışmaya katılan kişilerin görüşleri doğrudan alınmıştır. Veri Analizi Araştırmadan elde edilen nitel verilerin analizinde içerik analizinden yararlanılmıştır. Veri güvenirliğini sağlamak amacıyla katılımcılardan elde edilen veriler transkript edildikten sonra tekrar yüksek lisans öğrencilerine gönderilip teyit ettirilmiştir. Yüksek lisans öğrencilerinden alınan cevaplar araştırmacı tarafından düzenlenip belli temalar etrafında toplanmıştır. Daha sonra elde edilen nitel verilerin %50’ si uzmana gönderilip aynı işlemlerin yapılması istenmiştir. Çalışmada, ortak paydada birleşen kodlar kullanılmıştır. İçerik analizinde öncelikle birbirine benzeyen nitel veriler belli tema ve kategoriler altında düzenlenir daha sonra sebep-sonuç ilişkileri şeklinde yorumlanarak sonuçlara ulaşılır (Yıldırım & Şimşek, 2016). Bu araştırmada fen bilgisi eğitimi yüksek lisans öğrencilerinin soruları samimi ve içten bir şekilde cevaplamaları istenmiş ve vermiş oldukları cevaplar içerik analizi ile analiz edilmiştir (Marshall & Rossman, 2006). Verilerin analizinde, benzer yanıt veren katılımcı cevaplarından yola çıkarak aynı başlık altında toplanıp kodlar oluşturulmuştur. Son olarak veriler tablolaştırılıp yorumlanmıştır. Bilimsel etik çerçevesinde çalışmaya katılan kişilere kodlar verilmiştir (F1, F2, F3 gibi). EURASIA JOURNAL OF TEACHER EDUCATION, 2021, 2(3), 221-237 Veri Toplama Araçları Araştırma kapsamında fen bilgisi eğitimi yüksek lisans öğrencilerinin görüşlerini belirlemek amacıyla yarı yapılandırılmış görüşme formu kullanılmıştır. Yüksek lisans öğrencilerinin aşağıdaki soruları cevaplamaları istenmiştir. Tablo 1. Tablo 1. Katılımcıların STEM’ in bağlı olduğu disiplinlere ilişkin görüşleri Tablo 1. Katılımcıların STEM’ in bağlı olduğu disiplinlere ilişkin görüşleri Kodlar f Matematik 18 Mühendislik 18 Teknoloji 18 Fen Bilimleri 18 Fizik, Kimya, Biyoloji 2 Bilişim Teknoloileri 1 Disiplinlerarası 1 Disiplinlerarası Tablo 1 incelendiğinde katılımcıların tamamının (18 katılımcı) STEM ’i matematik, mühendislik, fen bilimleri ve teknolojiyle ilişkilendirdikleri görülmektedir. Katılımcıların bazıları (2 katılımcı) STEM’ i fizik, kimya ve biyolojiyle ilişkilendirmiştir. Katılımcılardan bir kişi ise (1 katılımcı) STEM ‘i disiplinlerarası kavramıyla ifade etmiştir. Tablo 1 incelendiğinde katılımcıların tamamının (18 katılımcı) STEM ’i matematik, mühendislik, fen bilimleri ve teknolojiyle ilişkilendirdikleri görülmektedir. Katılımcıların bazıları (2 katılımcı) STEM’ i fizik, kimya ve biyolojiyle ilişkilendirmiştir. Katılımcılardan bir kişi ise (1 katılımcı) STEM ‘i disiplinlerarası kavramıyla ifade etmiştir. Katılımcılardan F12'’nin görüşü şu şekildedir: Katılımcılardan F12'’nin görüşü şu şekildedir: ”STEM’in açılımı Fen bilimleri, Teknoloji, Mühendislik ve Matematiktir. Fen bilimleri, Teknoloji, Mühendislik ve Matematik disiplinlerinin bütüncül şekilde bir arada kullanılmasıyla öğretim gerçekleşmektedir. Fen ve Teknoloji kavramları birbirleriyle bütünsel olarak bağlantılı. Mesela fen alanında hipotez kurduk, hipotezi test etmek için teknolojiden yararlanırız veya teknolojide yeni bir icat için fen de elde edilen bilgiler kullanılır. Bilimsel çalışmalarda sayısal ifadeler olan veriler elde ettiğimizde çalışmamıza destekleyici ve ispat etmemize yardımcı olur. Bu sayede öğrenciler bu ispatla yola çıkarak gelişen teknolojiyi takip edebilirler. Bunlara ek olarak mühendislik sürecinde ise teknolojik ürünlerinin meydana gelmesinde Fen ve Matematik bilgileri kullanılır.’’ Katılımcılardan F18 ise görüşlerini şu şekilde belirtmiştir: ‘‘Fen Bilimleri (Fizik, Kimya, Biyoloji), Matematik, Teknoloji tasarım, Bilişim Teknolojileri gibi ana derslerin yanında multidisipliner olarak diğer tüm branşlar ile de ilişkilendirilebilir.’’ Katılımcıların 2. soru olan ‘‘STEM denildiğinde aklınıza neler gelmektedir?’’ sorusuna verdikleri cevaplar Tablo 2’de yer almaktadır. EURASIA JOURNAL OF TEACHER EDUCATION, 2021, 2(3), 221-237 225 GÜRBÜZ ET AL. GÜRBÜZ ET AL. Bulgular Bulgular Bulgular Bulgular Bu kısımda fen bilgisi eğitimi yüksek lisans öğrencilerine yöneltilen soruların cevapları yer almaktadır. Bu kısımda fen bilgisi eğitimi yüksek lisans öğrencilerine yöneltilen soruların cevapları yer almaktadır. Katılımcıların 1. soru olan ‘‘STEM hangi disiplinlerle ilişkilidir?’’ sorusuna verdikleri cevaplar Tablo 1’de yer almaktadır. Tablo 2. EURASIA JOURNAL OF TEACHER EDUCATION, 2021, 2(3), 221-237 Tablo 2. Katılımcıların STEM denildiğinde akıllarına gelenlere ilişkin görüşleri Kodlar f Teknoloji 14 Fen Bilimleri 11 Mühendislik 10 Matematik 9 Problem Çözme Becerisi 6 Disiplinlerarası Yaklaşım 6 Yaratıcı Düşünme 4 Ürün Oluşturma 3 VIEWS IN SCIENCE EDUCATION ON STEM EDUCATION 226 Proje Tabanlı Öğrenme 3 Eleştirel düşünme 2 Bilimsel Süreç Becerileri 1 Ekip Çalışması 1 Analitik Düşünme 1 Girişimcilik 1 Akıl Yürütme 1 Proje Tabanlı Öğrenme Eleştirel düşünme Bilimsel Süreç Becerileri Ekip Çalışması Analitik Düşünme Girişimcilik Akıl Yürütme Tablo 2 oluşturulurken katılımcıların tanım hakkında ifade ettiği terimler dikkate alınmıştır. Yüksek lisans öğrencilerinin verdikleri cevaplar dikkate alındığında, en fazla teknoloji kavramı üzerinde durdukları (14 katılımcı) daha sonra fen bilimleriyle ilişkilendirdikleri (11 katılımcı) görülmektedir. Katılımcıların en az ifade ettikleri terimler analitik düşünme (1 katılımcı), girişimcilik (1 katılımcı), akıl yürütme (1 katılımcı) ve ekip çalışması (1 katılımcı) olarak sıralanmaktadır. Katılımcılardan F6’nın görüşleri şu şekildedir: Katılımcılardan F6’nın görüşleri şu şekildedir: ‘‘Fen, teknoloji, mühendislik ve matematik kavramları geliyor. Bir araya gelen bu disiplinler, bireye çok boyutlu öğrenme sağlayabilir. Birey tüm bilgileri bütünleştirerek harmanlanmış bir bilgiye ulaşabilir. Bireylerin eleştirel düşünme, analitik düşünme ve disiplinler arası akıl yürütme becerilerini de artırabilir. STEM yaklaşımı bireylerin problem çözme becerilerine odaklı bir yaklaşım olduğunu düşünüyorum. Çünkü fen, teknoloji ve mühendisliğin hemen her alanında matematik kullanılır.’’ Katılımcıların 3. soru olan ‘‘STEM eğitiminin fen öğretimine katkısı olduğunu düşünüyor musunuz, kısaca açıklayınız?’’ sorusuna verdikleri cevaplar Tablo 3’de yer almaktadır. Tablo 3. Tablo 3. Katılımcıların STEM’ in Fen öğretimine katkısına ilişkin görüşleri Kodlar f Katkı sağlar 18 Günlük yaşamla ilişkili 7 Problem çözme becerisi 7 Kalıcılığı arttırır 6 Öğrenci aktiftir 4 Ürün oluşturulur 4 Yaratıcılığı arttırır 4 Uygulanabilirlik artar 3 Teknolojiyle ilişkili 3 Araştırmacılık 3 Disiplinlerarası etkileşim 3 Katılımcıların STEM’ in Fen öğretimine katkısına ilişkin görüşleri Tablo 3 incelendiğinde katılımcıların üçüncü soruya verdiği cevaplar görülmektedir. Katılımcıların tamamı (18 katılımcı) STEM eğitiminin fen öğretimi üzerinde olumlu katkısı olduğunu düşünmektedir. Katılımcılar STEM eğitiminin fen öğretimine katkısını sıralarken günlük yaşamla ilişkili olması (7 katılımcı), problem çözme becerisini geliştirdiği (7 katılımcı) ve dersin kalıcılığını arttırdığı (6 katılımcı) üzerinde durmuşlardır. Katılımcılardan F5’in görüşü şöyledir: Katılımcılardan F5’in görüşü şöyledir: ‘‘Evet düşünüyorum. Çünkü Fen Bilimleri soyut kavramları içerir. Öğrenciler tarafından anlaşılması zordur. Öğrenilmesi zor olan bu soyut kavramları kalıcı hale getirmek gerekir. Bu kavramları günlük hayatla ilişkilendirmek kalıcılığı sağlayacaktır. STEM eğitimiyle öğrenilen bilgiler kalıcı hale getirilebilir.’’ F13 ise görüşünü şöyle belirtmiştir: ‘‘STEM eğitiminin fen EURASIA JOURNAL OF TEACHER EDUCATION, 2021, 2(3), 221-237 227 GÜRBÜZ ET AL. öğretiminde katkısı olduğunu düşünüyorum. Çünkü Fen Bilimlerini de içeren bu yaklaşım sayesinde öğrenciler var olan bilgilerini hayata geçirme fırsatı buluyor. Çözüm üretiyor ve somut ürün ortaya çıkarıyor. Kalıcı öğrenmeyi sağlıyor. Derslere karşı ilgiyi artırıyor.’’ Katılımcıların 4. soru olan ‘‘Yüksek lisans eğitimi sırasında alanyazında STEM ile ilgili yapılmış güncel yayınları takip ediyor musunuz?’’ sorusuna verdikleri cevaplar Tablo 4’de yer almaktadır. Tablo 4 Tablo 4. Katılımcıların güncel yayınları takip etmelerine ilişkin görüşleri Katılımcıların güncel yayınları takip etmelerine ilişkin görüşleri Kodlar f Takip ediyorum 9 Takip etmiyorum 6 Kısmen 3 Tablo 4 incelendiğinde katılımcıların yarısı (9 katılımcı) STEM ile ilgili yapılmış güncel yayınları takip ettiklerini ifade etmişlerdir. Yüksek lisans eğitimini fen bilimleri üzerine yapmalarına rağmen katılımcıların, STEM ile ilgi alanyazında yeterli araştırma yapmadığı (6 katılımcı) ortaya çıkmaktadır. Bazı katılımcıların ise kısmen araştırma yaptıkları (3 katılımcı) görülmektedir. Katılımcılardan F8 görüşünü şu şekilde belirtmektedir: ‘‘Bu konu hakkında hiçbir yayın incelemedim en kısa zamanda araştırmalarıma başlayacağım.’’ F10’un görüşü ise ‘‘Evet ediyorum. Eğitim Bilimlerinde Akademik Yazım dersinde makale konum olarak probleme dayalı STEM etkinlikleri konusunu seçtim ve bunun üzerine tez ve makaleler okuyorum.’’ şeklindedir. Katılımcılardan F4: ‘‘Alanyazında STEM le ilgili yapılmış güncel yayınları takip etmeye çalışıyorum. İlgimi çeken ve eğitimini almak istediğim bir uygulama.’’ ifadelerini kullanmıştır. Katılımcıların 5. soru olan ‘‘Alanyazını incelediğinizde STEM ile ilgili yapılan çalışmaları yeterli buluyor musunuz?’’ sorusuna verdikleri cevaplar Tablo 5’de yer almaktadır. Tablo 5. Tablo 5. Katılımcıların çalışmaları yeterli bulup bulmadıklarına ilişkin görüşleri. Kodlar f Yeterli bulmuyorum 6 Eksiklikler mevcut 5 Daha fazla olmalı 5 Fikrim yok 4 Kısmen yeterli 3 Yeterli buluyorum 1 Katılımcıların çalışmaları yeterli bulup bulmadıklarına ilişkin görüşleri. Kısmen yeterli Yeterli buluyorum Yeterli buluyorum Tablo 5 incelendiğinde katılımcıların (6 katılımcı) alanyazındaki mevcut çalışmaları yeterli bulmadığı görülmektedir. Yüksek lisans öğrencileri alanyazını incelediklerinde, ilerleyen teknoloji etkisi ile mevcut çalışmaların sürekli artması gerektiğini dile getirmişlerdir (5 katılımcı). Katılımcılardan bazıları STEM ile ilgili yapılan çalışmalar hakkında bilgi sahibi olmadıklarını (4 katılımcı) ifade etmişlerdir. Katılımcılardan F10’a göre: ‘‘Tam olarak yeterli buluyorum demek yanlış olur. Basitçe, düşünürsek. Her gün teknoloji gelişmekte her yıl öğrencilerin yapısı tutumları vb. değişim içerisindeyken STEM le ilgili yapılan çalışmaların tam olması mümkün değildir.’’ bu şekildedir. F6’nın yorumu ise şu şekildedir: ‘‘Çok yeterli olduğunu düşünmüyorum. Henüz STEM in ne olduğunu bilmeyen çok kişi var. Ne olduğuna, nasıl ve nerelerde kullanılacağına, kullanımının ne gibi yararlar sağlayacağına dair alanyazına daha çok katkı yapılabilir.’’ VIEWS IN SCIENCE EDUCATION ON STEM EDUCATION Katılımcıların 6. soru olan ‘‘Yüksek lisans eğitimi süresince STEM eğitimine yönelik ders aldınız mı? STEM bilginizin yeterli olduğunu düşünüyor musunuz?’’ sorusuna verdikleri cevaplar Tablo 6’da yer almaktadır. Katılımcıların 6. soru olan ‘‘Yüksek lisans eğitimi süresince STEM eğitimine yönelik ders aldınız mı? STEM bilginizin yeterli olduğunu düşünüyor musunuz?’’ sorusuna verdikleri cevaplar Tablo 6’da yer almaktadır. EURASIA JOURNAL OF TEACHER EDUCATION, 2021, 2(3), 221-237 VIEWS IN SCIENCE EDUCATION ON STEM EDUCATION 228 Tablo 6. Katılımcıların STEM ile ilgili ders alıp almadıklarına ilişkin görüşleri Kodlar f Ders almadım 18 Yeterli bilgim yok 13 Eğitim almak isterim 4 Derslerde bahsedildi 2 Katılımcıların STEM ile ilgili ders alıp almadıklarına ilişkin görüşleri Tablo 6 incelendiğinde katılımcıların STEM eğitimine yönelik yeterli bilgi sahibi olmadıkları (13 katılımcı) ve hiçbirinin (18 katılımcı) yüksek lisans eğitimi süresince ders almadığı görülmektedir. Katılımcılardan bazıları ise ders almak istediklerini (4 katılımcı) ifade etmişlerdir. Katılımcılardan F10 fikirlerini şu şekilde belirtmiştir: Katılımcılardan F10 fikirlerini şu şekilde belirtmiştir: “Lisans eğitimimde STEM eğitimi aldım. Bir ders olarak adı STEM değildi fakat içeriği STEM olan bir ve birden fazla derste işledik. Pandemi araya girmeseydi projeler üretim sunum yapacaktık. Soruya dönecek olursak, yüksek lisans eğitiminde STEM e yönelik ders almadım. STEM konusunda yeterli olduğumu düşünmüyorum.’’ Yüksek lisansa başlamadan önce STEM eğitimi alan tek katılımcı mevcuttur. Katılımcılardan F12’nin görüşleri: ‘‘Eğitimimi yüksek lisans eğitiminden önce hizmet içi eğitim kapsamında 2 kur olarak aldım. Eğitim almama rağmen bilgilerimin yeterli olduğunu düşünmüyorum. Daha öğrenecek çok şeyin olduğunu biliyorum.” Katılımcıların 7. soru olan ‘‘Yüksek lisans tez konunuzun STEM eğitimi üzerine olmasını tercih eder misiniz?’’ sorusuna verdikleri cevaplar Tablo 7’de yer almaktadır. Tablo 7. ıların tez konularında STEM eğitimini tercih etmeye ilişkin görüşleri Katılımcıların tez konularında STEM eğitimini tercih etmeye ilişkin görüşleri Kodlar f Tercih edebilirim 17 Faydalı olur 3 Popüler bir konu 2 Güncel 2 Orijinal bir konu 1 Kararsızım 1 Tercih etmem 1 Tablo 7 incelendiğinde katılımcıların büyük bir kısmının (17 katılımcı) tez konusu hakkında STEM eğitimi üzerine çalışma yapmak istediği görülmektedir. Katılımcılardan bazıları ise tez konularının STEM eğitimi üzerine olmasını popüler (2 katılımcı), orijinal (1 katılımcı) ve güncel bir konu (2 katılımcı) olmasıyla ilişkilendirmiştir. Yüksek lisans öğrencilerinden F1 fikirlerini şu şekilde dile getirmiştir: ‘“Yüksek lisans tezi için tercih edebilirdim güncel ve popüler bir konu olması meslek hayatımda uygulanabilirliği açısından kendime faydalı olabileceğini düşünüyorum. Yapılacak araştırmada kaynak tarama kısmında zorlanmayacağım içinde tercih edebilirdim.’’ F3’ün görüşleri: ‘‘STEM uzun yıllardır çalışılan bir yaklaşım ve günümüzde hala popülaritesini koruyor. STEM ile ilgili iddialı bir konum olursa tezde çalışabilirim.’’ F16 STEM’in kendi gelişimi için olumlu olacağını şu sözlerle belirtmiştir: ‘‘Tez konum STEM eğitimi üzerine olsun isterim. Çünkü fen bilimleri öğretmeniyim. EURASIA JOURNAL OF TEACHER EDUCATION, 2021, 2(3), 221-237 229 GÜRBÜZ ET AL. GÜRBÜZ ET AL. STEM disiplinlerinden biri fen bilimleri. Yani kendi branşım. Kendi branşımı teknoloji, mühendislik ve matematikle süslemek hem kendi gelişimim açısından hem de çocuklara bir şeyler katmak açısından faydalı olacağını düşünüyorum.’’ STEM disiplinlerinden biri fen bilimleri. Yani kendi branşım. Kendi branşımı teknoloji, mühendislik ve matematikle süslemek hem kendi gelişimim açısından hem de çocuklara bir şeyler katmak açısından faydalı olacağını düşünüyorum.’’ Katılımcıların 8. soru olan ‘‘Öğretmenlik uygulaması dersi kapsamındaki uygulamalarınızda veya öğretmenlik mesleğinizde STEM etkinliklerini kullanarak neler yaptınız? STEM etkinliklerini kullanmadıysanız neden kullanmadınız?’’ sorusuna verdikleri cevaplar Tablo 8’de yer almaktadır. Tablo 8. o 8. ımcıların hangi STEM etkinliklerini kullandıklarına ilişkin görüşleri Tablo 8. Katılımcıların hangi STEM etkinliklerini kullandıklarına ilişkin görüşleri Kodlar f Uygulamadım 12 Eğitimini almadım 4 Zaman kısıtlı 2 Eksik materyallerden dolayı kullanamadım 2 Isı yalıtımı 1 Maddenin tanecikli yapı modeli 1 Çeşitli etkinlikler 1 Günlük yaşamdan örnekler 1 Materyal tasarımı 1 Ürün oluşturdum 1 Yaratıcı fikirler çıktı 1 Solunum Sistemi 1 Pandemiden dolayı kullanamadım 1 Robot 1 Mesleği yapmadığımdan dolayı 1 Aktif ders sürecinde olmadığım için kullanmadım 1 Tablo 8 incelendiğinde katılımcıların çoğunun (12 katılımcı) derslerde STEM etkinliklerini kullanmadığı görülmektedir. Katılımcılar bunun nedeni olarak farklı farklı sebepler sunmuştur. Bunlardan bazıları şunlardır; STEM etkinliği yaptırmanın müfredattaki zaman akışını olumsuz etkilediği (2 katılımcı), yeterli materyal olmaması (2 katılımcı) ve eğitimini almadıklarından dolayı (4 katılımcı) kullanamadıklarını belirtmişlerdir. Sınıf içerisinde STEM etkinliklerine yer vermeyen katılımcılardan biri olan F14 şu şekilde açıklama yapmıştır: ‘‘Öğretmenlik mesleğinde STEM etkinliklerini kullanamadım sebebi aldığım eğitimden sonra önce tatillerin sonra pandemi dönemine girmiş olmamız. Bütün bunlar olmasaydı yine de kullanmak zor olabilirdi. Hem ekipmanların olmaması hem de müfredata ayrılan zamanın ancak kendine yetebilmesi.’’ STEM etkinliğini sınıfında uygulayan F2’nin görüşü ise: ‘‘Solunum sistemi çalışma modeli, sıvı basıncından (Pascal prensibinden) yararlanarak manuel kontrol edilebilen robot vb. Etkinlikler’’ bu şekildedir. Yine STEM etkinliğini sınıfında uygulayan katılımcılardan F5 görüşünü şöyle belirtmiştir: ‘‘Lisans eğitiminde iken Öğretim Teknolojileri Materyal Tasarımı dersinde atık maddeleri kullanarak 6.sınıf Fen Bilimleri müfredatındaki Maddenin Tanecikli Yapısı konusunu içeren, tanecik hareketlerini gösteren bir materyal tasarlamıştım. Bu materyal öğrencilerin anlamada güçlük çektiği soyut kavramları somutlaştırarak daha kolay anlamalarını sağlamak amaçlı idi. Ayrıca öğrencilerin derse katılımını arttırarak öğrenmeyi de kalıcı hale getirmekte idi.’’ ‘‘Lisans eğitiminde iken Öğretim Teknolojileri Materyal Tasarımı dersinde atık maddeleri kullanarak 6.sınıf Fen Bilimleri müfredatındaki Maddenin Tanecikli Yapısı konusunu içeren, tanecik hareketlerini gösteren bir materyal tasarlamıştım. Bu materyal öğrencilerin anlamada güçlük çektiği soyut kavramları somutlaştırarak daha kolay anlamalarını sağlamak amaçlı idi. Ayrıca öğrencilerin derse katılımını arttırarak öğrenmeyi de kalıcı hale getirmekte idi.’’ Katılımcıların 9. soru olan ‘‘Fen bilimleri dersinde STEM etkinlikleri yaptırmanın avantaj ve dezavantajları nelerdir?’’ sorusuna verdikleri cevaplar Tablo 9’da yer almaktadır. Katılımcıların 9. soru olan ‘‘Fen bilimleri dersinde STEM etkinlikleri yaptırmanın avantaj ve dezavantajları nelerdir?’’ sorusuna verdikleri cevaplar Tablo 9’da yer almaktadır. EURASIA JOURNAL OF TEACHER EDUCATION, 2021, 2(3), 221-237 VIEWS IN SCIENCE EDUCATION ON STEM EDUCATION 230 Tablo 9. Tablo 8. Katılımcıların STEM yaptırmanın avantaj ve dezavantajlarına ilişkin görüşleri Kodlar f Avantajları Problem çözme becerisi kazanma 8 Öğrenci aktifliği 6 Kalıcı öğrenmeler 5 Farklı bakış açıları kazandırma 5 Derse istekli olma 5 Derse olumlu tutum oluşturma 5 Bilimsel süreç becerilerini kullanma 3 Motor becerisi kazandırma 3 Eleştirel düşünme becesiri kazanma 2 İşbirliği 2 Yaratıcı düşünme 2 Bilgim yok 2 Soyut kavramları somutlaştırma 1 İnovasyon 1 Dezavantajları Zaman sıkıntısı 8 Ekonomik değil 6 Öğrenci sıkılabilir 4 Materyal eksikliği 3 Öğretmenin bilgi eksikliği 3 Bilgim yok 2 Dezavantajları Materyal eksikliği Öğretmenin bilgi eksikliği Tablo 9 incelendiğinde, yüksek lisans öğrencilerinin fen bilimleri dersinde STEM etkinliği yaptırmanın avantaj ve dezavantajlarına verdikleri cevaplardan oluşan kodlar tablodaki gibidir. Katılımcılar STEM etkinliklerinin öğrencinin yaratıcı düşünme (2 katılımcı), derse karşı olumlu tutum sergileme (5 katılımcı) ve problem çözme becerisi geliştirme (8 katılımcı) gibi olumlu yönlerinden bahsetmişlerdir. Bunun yanında STEM etkinliklerinin uygulanma sürecinde öğrencilerin sıkılabileceğini (4 katılımcı) ve ekonomik olmadığını (6 katılımcı) dile getirmişlerdir. Katılımcılardan F2’nin görüşleri şu şekildedir: ‘‘Öğrencilerde kalıcı öğrenmeler sağlaması, derse öğrencinin ilgisinin kolayca çekilebilmesi, öğrenciler ince motor kas becerileri geliştirmesi olumlu yönleri; yeterli zaman bulunamaması, kullanılacak malzemelerinin tespiti vb. yanları olumsuz olarak görülebilir.’’ Katılımcılardan F3’ün soru hakkındaki düşüncesi şu şekildedir: ‘‘Öğretimin verimini artırır. Öğrenciyi süreç boyunca aktif tutar. Öğrenciye farklı bakış açıları kazandırır. İşbirliğini kuvvetlendirir. Ekonomik (zaman ve para) değildir.’’ Katılımcıların 10. soru olan ‘‘Fen bilimleri dersinde STEM etkinliklerini arttırmak için hangi çalışmalar yapılmalıdır?’’ sorusuna verdikleri cevaplar Tablo 10’da yer almaktadır. EURASIA JOURNAL OF TEACHER EDUCATION, 2021, 2(3), 221-237 231 GÜRBÜZ ET AL. Tablo 10. Katılımcıların STEM’ i arttırmak için yapılması gerekenlere ilişkin görüşleri Kodlar f Etkinlikler arttırılmalı 9 Nitelikli öğretmen yetiştirilmeli 8 Materyal eksikliği giderilmeli 8 Süreç değerlendirmesi yapılmalı 3 Ders planı yapılmalı 3 Ders somutlaştırılmalı 2 Simülasyon kullanımı arttırılmalı 2 Hayatilik 2 Disiplinlerarası yaklaşım 2 Yaşama aktarım 1 Bilim şenlikleri arttırılmalı 1 Seçmeli ders yapılabilir 1 o 10. ımcıların STEM’ i arttırmak için yapılması gerekenlere ilişkin görüşleri Seçmeli ders yapılabilir Tablo 10 incelendiğinde katılımcıların, fen bilimleri dersinde STEM etkinliklerini arttırmak için öğretmenlerin bu konuda eğitim alarak daha yeterli hâle gelmeleri (8 katılımcı) ve okullarda yaşanan materyal sıkıntılarının giderilmesi gerektiğinin (8 katılımcı) üzerinde durmuşlardır. Katılımcıların yarısı ise etkinliklerin arttırılması (9 katılımcı) gerektiğini ifade etmişlerdir. Tablo 8. Katılımcılardan F9’un görüşü şu şekildedir: ‘‘STEM etkinlikleri arttırmak amacı ile model simülasyon sayıları arttırılmalı STEM etkinlik programı günlük hayata olan etkileri daha çok ön plana çıkmalı’’ Katılımcılardan F11 ise fikrini şu şekilde açıklamıştır: ‘‘Gerekli araç gereç temin edildiğinde bir birim oluşturularak öğretmenlere teknik destek sağlanabilir. Bu eğitimler ile fen bilimleri öğretmenleri STEM’i sınıflarında uygulamaları konusunda cesaretlendirilebilirler.’’ Katılımcılardan F12’nin görüşü: ‘‘Öncelikle öğretmenlerin iyi bir STEM eğitimi alması gerekir. Daha sonra çocuklara STEM hakkında bilgilendirme yapılmalı. Okula gerekli ekipmanlar tedarik edilmeli gerekirse laboratuvar gibi mini STEM odası kurulmalı. Bu biraz pahalı olabileceği için Bayburt’taki STEM merkezinden randevu oluşturulup belli aralıklarla gidilmesi sağlanabilir. Okulda STEM kulübü de kurulabilir. Seçmeli ders olarak da eklenebilir.’’ VIEWS IN SCIENCE EDUCATION ON STEM EDUCATION 232 Benzer (2018) 7E öğrenme modeli merkezli STEM etkinliğine dayalı öğretim uygulamalarının akademik başarıya etkisini araştırmışlardır. STEM etkinliğine dayalı öğretimde öğrencilerin akademik başarısında artış olduğu tespit edilmiştir. Yine Yıldırım ve Selvi (2017) ortaokul öğrencileriyle STEM uygulamaları ve tam öğrenmenin öğrencilerin akademik başarılarına etkisini araştırdıkları çalışmada STEM uygulamaları ve tam öğrenmenin 7.sınıf öğrencilerin akademik başarılarını artırdığını tespit etmişlerdir. Katılımcıların çoğu yüksek lisans eğitimleri süresince alanyazında STEM ile ilgili yapılmış çalışmaları takip ettiğini veya kısmen takip ettiğini dile getirmiştir. Katılımcıların bazıları ise STEM ile ilgili yapılan çalışmaları takip etmediğini ifade etmiştir. Yüksek lisansta verilen dersler kapsamında STEM eğitimine olan ilgi ve merak daha da genişletilebilir. Bu bağlamda ders alma döneminde olan öğrencilere STEM eğitimini de içine alan güncel alanyazın tarama araştırmaları verildiğinde STEM’ e karşı bakış açısının değiştiği sonucuna ulaşılmıştır. Alanyazındaki mevcut çalışmaların yetersiz olduğu, gelişen teknoloji ve hayat standartları neticesinde bu gibi çalışmaların arttırılması gerektiği sonucuna ulaşılmıştır. Alanyazın incelendiğinde STEM ile ilgili gerek nicel gerekse nitel birçok çalışma mevcuttur ancak yeni bir yaklaşım olduğu için geliştirilmesi ve uygulanabilirliği arttırılmalıdır (Ergün & Balçın, 2018; Kaya, Şahin & Uz-Özkılıç, 2020; Yıldırım & Selvi, 2017). Katılımcılar yüksek lisans ders alma dönemlerinde STEM ile ilgili ders almadıklarını dile getirmişler ayrıca bu konu hakkında yeterli bilgiye sahip olmadıklarını belirtmişlerdir. Ancak eksikliklerinin giderilmesi için eğitim almaya gönüllü olduklarını ifade etmişlerdir. Katılımcılardan birkaçı yüksek lisansa başlamadan önce hizmet içi seminerlerden dolayı bilgi sahibi olduğunu belirtmiştir. Aynı zamanda katılımcılardan bazıları STEM eğitiminin ders olarak verilmediği ancak derslerde bahsedildiği ve ders veren öğretmenler tarafından öneminin derslerde vurgulandığını belirtmişlerdir. Bu bağlamda yüksek lisansta ders veren öğretmenlerin STEM eğitiminin faydalarını ve nasıl uygulanması gerektiğini öğrencilerine sunmalı ve onları STEM eğitimi konusunda yönlendirmelidir. Elmalı ve Kıyıcı (2017)’ nın çalışması incelendiğinde STEM eğitiminin öğrenciler üzerinde olumlu etkiler bıraktığı, fen bilimleri dersi akademik başarısını arttırdığı, işbirlikli öğrenmeyi geliştirdiği ve derse karşı olumlu tutum oluşturduğu görülmektedir. Bu bağlamda STEM etkinliklerini içine alan çalışmaların, farklı seviyedeki öğrencilerde birçok olumlu etki bıraktığı sonucuna ulaşılmaktadır. Bu araştırmanın sonucunda da yapılan çalışmalarla paralellik gösterecek şekilde yüksek lisans döneminde STEM ile ilgili ders almanın sınıf içinde yaptırılan etkinlikleri arttırabileceği sonucuna ulaşılmıştır. Katılımcıların yüksek lisans tez konusu seçmede STEM ile ilgili konuların tercih edilip edilmeyeceği sorulduğunda ise yüksek oranda tercih edeceklerini belirtmişlerdir. Tez konusu seçme konusunda özgün, güncel ve orijinal bir fikir oluşturmanın önemli olduğunu dile getirmişlerdir. Bu bağlamda STEM ile ilgili konu seçiminin alanyazına daha çok katkı sağlayacağı belirlenmiştir. Tartışma ve Sonuç Katılımcılar, yönlendirilen ilk iki soru ışığında STEM’in hangi disiplinlerle ilişkili olduğunu ve STEM denildiğinde akıllarına nelerin geldiğini cevaplamışlardır. Bu cevaplar göz önüne alındığında katılımcıların STEM’i açıklamada fen bilimi, mühendislik, matematik, teknoloji gibi kavramları kullandıkları görülmüştür. Ayrıca katılımcılar fen bilimleri dersinde STEM etkinliklerine yer vermenin gerekli olduğunu savunmuşlardır. Derslerde yapılacak etkinlikler sayesinde öğrencilerde yaratıcı düşünme, problem çözme becerisi, eleştirel düşünme, akıl yürütme, işbirlikli öğrenme ve derse karşı olumlu tutum geliştirme gibi özelliklerin artacağını dile getirmişlerdir. Derslerde kullanılan STEM etkinlikleri sayesinde konunun akılda kalıcılığında artış olacağı düşünülmektedir. Bu bağlamda öğrencilerin fen bilimleri dersi akademik başarısında artış olacağı söylenebilir. Aynı zamanda fazlaca soyut kavram içeren fen bilimleri dersinde, kavramların somutlaştırılması konusunda etkili bir yöntem olduğu sonucuna ulaşılmıştır. Alanyazın incelendiğinde bununla paralellik gösteren çalışmalar mevcuttur. Birçok çalışmada STEM etkinliklerinin kullanılması sonucunda öğrencilerde 21. yüzyıl becerilerinin geliştiği görülmüştür (Bakırcı & Kutlu, 2018; Eroğlu & Bektaş, 2016; Güldemir & Çınar, 2017; Güven, Selvi & Benzer, 2018; Yıldırım, 2017; Yıldırım & Altun, 2015; Yıldırım & Selvi, 2017). Güven, Selvi ve EURASIA JOURNAL OF TEACHER EDUCATION, 2021, 2(3), 221-237 VIEWS IN SCIENCE EDUCATION ON STEM EDUCATION VIEWS IN SCIENCE EDUCATION ON STEM EDUCATION Alanyazın incelendiğinde özellikle fen bilimleri ders konularının soyut kavramlar içermesinden dolayı öğrencilerin bu dersi anlamada güçlük yaşadığı bilinmektedir. Fen bilimleri dersinin anlaşılmasında öğrencilerin olaya aktif olarak dahil edilmesi, bilimsel süreç becerilerini kullanmaları öğrencilerin gelişimlerini olumlu yönde arttırdığı yapılan çalışmalarda tespit edilmiştir (Bakırcı & Kutlu, 2018; Eroğlu & Bektaş, 2016). Katılımcılara öğretmenlik uygulaması kapsamında veya öğretmenlik mesleklerinde STEM etkinliklerini kullanarak neler yaptıkları sorulduğunda anlamlı ölçüde uygulama yapmadıkları tespit edilmiştir (Tablo 8). Yüksek lisans öğrencilerinin uygulama yapamama sebepleri arasında yetersiz materyal, STEM için uygun olmayan sınıf ortamı ve zamanın kısıtlı olması sonuçlarına ulaşılmıştır. Siew, Amir ve Chong (2015) yapmış oldukları çalışmada STEM etkinliklerinin çok fazla zaman alabileceğini, maliyet açısından fazla olduğunu ve materyal bulmada sıkıntı oluşabileceğini belirtmişlerdir. Katılımcılardan alınan cevaplar bu çalışmayı destekler niteliktedir. Bir katılımcı ise pandemiden hemen önce göreve başladığından dolayı STEM etkinliklerini uygulayamadığından bahsetmiştir. Pandemi döneminde uzunca bir süre dersler uzaktan eğitim yoluyla işlendiğinden dolayı öğrencilerle etkileşimli bir şekilde gözlem ve deney yapmak zorlaşmıştır. Öğrencinin derse aktif olarak katılamaması ve öğretmen ile öğrenci arasında EURASIA JOURNAL OF TEACHER EDUCATION, 2021, 2(3), 221-237 233 GÜRBÜZ ET AL. GÜRBÜZ ET AL. yaşanan kopukluklardan dolayı STEM etkinliklerinin kullanımı sekteye uğramıştır (Benzer & Akkaya, 2021). Buradan hareketle uzaktan eğitim sürecinde öğretmenlerin online derslerde STEM etkinlikleri yaptırmasının zor olduğu sonucuna ulaşılmıştır. Nitekim tüm bu olumsuzluklara rağmen STEM etkinlikleri yaptıran katılımcılar da mevcuttur (Tablo 8). yaşanan kopukluklardan dolayı STEM etkinliklerinin kullanımı sekteye uğramıştır (Benzer & Akkaya, 2021). Buradan hareketle uzaktan eğitim sürecinde öğretmenlerin online derslerde STEM etkinlikleri yaptırmasının zor olduğu sonucuna ulaşılmıştır. Nitekim tüm bu olumsuzluklara rağmen STEM etkinlikleri yaptıran katılımcılar da mevcuttur (Tablo 8). Fen bilgisi eğitimi yüksek lisans öğrencilerine STEM etkinlikleri yaptırmanın avantaj ve dezavantajları sorulmuştur. Katılımcıların büyük bir kısmı avantaj olarak problem çözme becerisine olumlu katkıda bulunacağını söylemiştir. Bu bağlamda STEM etkinliklerinin kalıcı öğrenmeleri arttıracağı, öğrenciye çok yönlü düşünme becerisi katacağı, öğrenciyi derste aktif kılacağı ve öğrencinin derse karşı olumlu tutum sergileyeceği sonucuna ulaşılmıştır. Altan, Yamak ve Kırıkkaya (2016) çalışmasında, öğretmen adaylarının STEM etkinliklerinin yaparak yaşayarak öğrenmeyi arttırdığını, kalıcılığı sağladığını ve derse karşı motivasyonu olumlu yönde etkilediğini ifade etmişlerdir. Ayrıca Sümen ve Çalışıcı (2016) yaptıkları çalışma sonucunda derste STEM etkinlikleri yaptırmanın aktif katılım oluşturduğundan dolayı derslerin daha verimli geçtiğini belirtmişlerdir. Bu çalışmadan elde edilen bulgular da alanyazında var olan çalışmaları destekler niteliktedir. Derste STEM etkinlikleri yaptırmanın olumsuz yönleri ise zaman ve materyal sıkıntısı, ekonomik olmaması, öğretmenin bilgi yetersizliği ve öğrencinin dersten sıkılıp kopması olarak belirlenmiştir. VIEWS IN SCIENCE EDUCATION ON STEM EDUCATION Fen bilgisi eğitimi yüksek lisans öğrencilerine son olarak STEM etkinliklerini arttırmak için hangi çalışmalar yapılmalıdır sorusu yöneltilmiştir. Katılımcılardan elde edilen cevaplar doğrultusunda sınıf içinde yapılacak etkinliklerin arttırılması ve STEM eğitimi hakkında yeterli bilgiye sahip öğretmenler yetiştirilmesi gerektiği sonucuna ulaşılmıştır (Tablo 10). Ayrıca katılımcılar öğretmenlerin derslerinde simülasyon kullanmaları gerektiğini savunmuşlardır. Değerlendirmenin sadece ürün odaklı olmaması, sürece yayılan bir değerlendirme olması gerektiği, bu sayede öğrencilere daha sağlıklı dönütler verileceği belirlenmiştir. Alanyazın incelendiğinde STEM yaklaşımının yeni bir uygulama olması ve ayrı bir ders olarak öğretmenlere lisans eğitiminde verilmemesinden dolayı çeşitli olumsuzluklara yol açtığı sonucuna ulaşılmıştır. Ayrıca öğretmenlerin sınıf içinde yaptırılan STEM etkinliklerinin sonuç yerine süreç odaklı değerlendirme olması üzerinde durulmuştur (Bakırcı & Karışan, 2018; Bakırcı & Kutlu, 2018; İdin, 2017). Öneriler Araştırmaya katılan fen bilimleri yüksek lisans öğrencilerinin STEM eğitimi hakkındaki görüşleri dikkate alındığında, öğretmenlere yeterli seviyede STEM eğitimi verilmesinin gerekli olduğu görülmüştür. Bundan dolayı eğitim fakültelerinin yeterli seviyede STEM eğitimi verebilmesi için müfredatlarında yeniliğe gitmeleri önerilebilir. Çünkü STEM etkinliklerinin uygulayıcısı öğretmenlerdir ve yeterince alan bilgisine sahip olmayan öğretmenler verimli bir şekilde ders işleyemez. Yüksek lisans eğitimi veren enstitülerin STEM eğitimi ile ilgili ders vermeleri önerilebilir. Tez yazma döneminde olan yüksek lisans öğrencilerinin, STEM eğitimi ile ilgili tez çalışması yapmaları teşvik edilmelidir. Bu sayede STEM eğitimi ile ilgili alanyazına daha çok katkı sağlanabilir. Ayrıca yüksek lisans eğitimi sırasında öğretmenlerin, öğrencilerine güncel alanyazın taraması yaptırmaları önerilebilir. Sınıf içerisinde STEM temelli ders etkinliklerinin sayısının arttırılması önerilebilir. Bu sayede oldukça fazla soyut kavram içeren fen bilimleri dersinin anlaşılma seviyesi arttırılabilir. Derslerinde STEM etkinliklerini uygulamak isteyen öğretmenlerin süreç değerlendirmesine önem vermesi ve materyal kullanımını arttırması önerilebilir. STEM etkinliğini uygulayabilmek için yeterli zaman bulamadıklarını ifade eden katılımcıların görüşleri dikkate alındığında, mevcut fen bilimleri müfredatının STEM uygulamalarıyla entegre bir şekilde yeniden düzeltilip dizayn edilmesinin gerekliliği görülmektedir. Bu çalışma 2021-2022 yılında güz döneminde fen bilgisi eğitimi yüksek lisans öğrencilerinden oluşan 18 öğretmenle gerçekleştirilmiştir. STEM eğitimi hakkında görüşlerin inceleneceği farklı örneklem gruplarıyla çalışmalar yapılabilir. EURASIA JOURNAL OF TEACHER EDUCATION, 2021, 2(3), 221-237 EURASIA JOURNAL OF TEACHER EDUCATION, 2021, 2(3), 221-237 VIEWS IN SCIENCE EDUCATION ON STEM EDUCATION 234 Kaynakça Alagöz, S., & Sözen, E. (2021). Sınıf öğretmenlerinin STEM eğitimi hakkındaki görüşleri. Üçüncü Sektör Sosyal Ekonomi Dergisi, 56(2), 1245-1266. https://doi.org/10.15659/3.sektor- sosyal-ekonomi.21.06.1576 Aslan, F., & Bektaş, O. (2019). Fen bilgisi öğretmen adaylarının STEM uygulamaları hakkındaki görüşlerinin belirlenmesi. Maarif Mektepleri Uluslararası Eğitim Bilimleri Dergisi, 3(2), 17- 50. https://doi.org/10.46762/mamulebd.646318 Bakırcı, H., & Karışan, D. (2018). Investigating the preservice primary school, mathematics and science teachers’ stem awareness. Journal of Education and Training Studies, 6(1), 32- 42. https://doi.org/10.11114/jets.v6i1.2807 Bakırcı, H., & Kutlu, E. (2018). Fen bilimleri öğretmenlerinin FeTeMM yaklaşımı hakkındaki görüşlerinin belirlenmesi. Türk Bilgisayar ve Matematik Eğitimi Dergisi, 9(2), 367-389. https://doi.org/10.16949/turkbilmat.417939 Benzer, S., & Akkaya, M. M. (2021). Pandemi sürecinde fen bilimleri alanında uzaktan eğitim. SBedergi, 5(8), 19-46. https://dx.doi.org/10.29228/sbe.49094 Bozkurt-Altan, E., Yamak, H., & Buluş-Kırıkkaya, E. (2016). Hizmet öncesi öğretmen eğitiminde FeTeMM eğitimi uygulamaları: Tasarım temelli fen eğitimi. Trakya Üniversitesi Eğitim Fakültesi Dergisi, 6(2), 212-232. Ceylan, S. (2021). Stem ve eğitimde kullanımına yönelik yapılan lisansüstü çalışmaların incelenmesi. Trakya Eğitim Dergisi, 11(2), 820-837. https://doi.org/10.24315/tred.700713 Creswell, J. W. (2003). Research design: Qualitative, quantitative, and mixed methods approaches. Thousand Oaks, CA: Sage. Elmalı, Ş., & Balkan-Kıyıcı, F. (2017). Türkiye’de yayınlanmış FeTeMM eğitimi ile ilgili çalışmaların incelenmesi. Sakarya University Journal of Education, 7(3), 684-696. https://doi.org/10.19126/suje.322791 Ergün, A., & Balçın, M. D. (2018). Perceptions and attitudes of secondary school students towards engineers and engineering. Journal of Education and Practice, 9(10), 90-106 Eroğlu, S., & Bektaş, O. (2016). STEM eğitimi almış fen bilimleri öğretmenlerinin stem temelli ders etkinlikleri hakkındaki görüşleri. Eğitimde Nitel Araştırmalar Dergisi, 4(3), 43-67. https://doi.org/10.14689/issn.2148-2624.1.4c3s3m Gonzalez, H. B., & Kuenzi, J. J. (2012). Science, technology, engineering, and mathematics (STEM) education: A primer. Congressional Research Service, Library of Congress. Güldemir, S., & Çınar, S. (2017). Fen bilimleri öğretmenleri ve ortaokul öğrencilerinin stem etkinlikleri hakkındaki görüşleri. ULEAD 2017 Annual Congress: ICRE. Güven, Ç., Selvi, M., & Benzer, S. (2018). 7E öğrenme modeli merkezli STEM etkinliğine dayalı öğretim uygulamalarının akademik başarıya etkisi. Anemon Muş Alparslan Üniversitesi Sosyal Bilimler Dergisi, 6(STEMES’18), 73-80. https://doi.org/10.18506/anemon.463812 Herdem, K., & Ünal, İ. (2018). STEM eğitimi üzerine yapılan çalışmaların analizi: Bir meta-sentez çalışması. Marmara Üniversitesi Atatürk Eğitim Fakültesi Eğitim Bilimleri Dergisi, 48(48), 145-163 https://doi.org/10.15285/maruaebd.345486 İdin, Ş. (2017). Örnek ve uygulama destekli fen öğretiminde disiplinler arası beceri etkileşimi E. Kandemir (Ed.), STEM yaklaşımı ve eğitime yansımaları içinde (s. 255-282). Pegem Akademi. EURASIA JOURNAL OF TEACHER EDUCATION, 2021, 2(3), 221-237 EURASIA JOURNAL OF TEACHER EDUCATION, 2021, 2(3), 221-237 235 GÜRBÜZ ET AL. GÜRBÜZ ET AL. Kaya, G., Şahin, E., & Uz-Özkılıç, R. (2020). Fen bilimleri öğretmenlerinin stem eğitimi ve uygulamaları hakkındaki görüşlerinin belirlenmesi (Bursa ili örneği). Uluslararası İnsan ve Sanat Araştırmaları Dergisi, 5(6), 100-113. Marshall, C., & Rossman, G.B. (2006). Designing qualitative research (4th ed.). Sage Publications. Milli Eğitim Bakanlığı. (2018). Fen bilimleri dersi (3, 4, 5, 6, 7 ve 8. sınıflar) öğretim programı. Milli Eğitim Bakanlığı. https://mufredat.meb.gov.tr/Dosyalar/201812312311937- FEN%20B%C4%B0L%C4%B0MLER%C4%B0%20%C3%96%C4%9ERET%C4%B0M%20 PROGRAMI2018.pdf Milli Eğitim Bakanlığı. (2020). TİMSS 2019 Türkiye Ön Raporu. Milli Eğitim Bakanlığı. http://odsgm.meb.gov.tr/meb_iys_dosyalar/2020_12/10175514_TIMSS_2019_Turkiye_O n_Raporu_.pdf Sümen, Ö. Ö., & Çalışıcı, H. (2016). Pre-service teachers' mind maps and opinions on STEM education implemented in an environmental literacy course. Educational Sciences: Theory and Practice, 16(2), 459-476. https://doi.org/10.12738/estp.2016.2.0166 Siew, N. M., Amir, N., & Chong, C. L. (2015). The perceptions of pre-service and inservice teachers regarding a project-based STEM approach to teaching science. Springer Plus, 4(8), 1-20. https://doi.org/10.1186/2193-1801-4-8 Türk Sanayicileri ve İş Adamları Derneği. (2017). 2023’e doğru Türkiye’ de STEM gereksinimi. https://tusiad.org/tr/yayinlar/raporlar/item/9735-2023-e-dog-ru-tu-rkiye-de-stem- gereksinimi Yıldırım, A., & Şimşek, H. (2016). Sosyal bilimlerde nitel araştırma yöntemleri (10.baskı). Seçkin Yayıncılık. Yıldırım, B. (2018). STEM uygulamalarına yönelik öğretmen görüşlerinin incelenmesi. Eğitim Kuram ve Uygulama Araştırmaları Dergisi, 4(1), 42-53. Yıldırım, B., & Altun Y. (2015). STEM eğitim ve mühendislik uygulamalarının fen bilgisi laboratuar dersindeki etkilerinin incelenmesi. El-Cezerî Fen ve Mühendislik Dergisi, 2(2), 28-40. https://doi.org/10.31202/ecjse.67132 Yıldırım, B., & Selvi, M. (2017). STEM uygulamaları ve tam öğrenmenin etkileri üzerine deneysel bir çalışma. Eğitimde Kuram ve Uygulama Journal of Theory and Practice in Education, 13(2), 183-210 https://doi.org/10.17244/eku.310143 Yıldırım, B., & Selvi, M. (2018). Ortaokul öğrencilerinin STEM uygulamalarına yönelik görüşlerinin incelenmesi. Anemon Muş Alparslan Üniversitesi Sosyal Bilimler Dergisi, 6(STEMES’18), 47-54. https://doi.org/10.18506/anemon.471037 Yıldırım, B., & Türk, C. (2018). Sınıf öğretmeni adaylarının STEM eğitimine yönelik görüşleri: uygulamalı bir çalışma. Trakya Üniversitesi Eğitim Fakültesi Dergisi, 8(2), 195-213. https://doi.org/10.24315/trkefd.310112 Yıldırım, P. (2017). Fen teknoloji mühendislik ve matematik (STEM) entegrasyonuna ilişkin nitel bir çalışma. Atatürk Üniversitesi Kazım Karabekir Eğitim Fakültesi Dergisi, (35), 31-55. Extended Abstract Extended Abstract Results Science education graduate students were asked about the advantages and disadvantages of having STEM activities. Most of the participants stated that it would contribute positively to their problem-solving skills as an advantage. In this context, it has been concluded that STEM activities will increase permanent learning, add multi-dimensional thinking skills to the student, make the student active in the lesson and show a positive attitude towards the lesson. At study of Altan, Yamak, and Kırıkkaya (2016) stated that pre-service teachers' STEM activities increase learning by doing, provide permanence, and positively affect motivation towards the lesson. In addition, Sümen and Çalışıcı (2016) stated that as a result of their study, having STEM activities in the lesson creates active participation in the lessons, so the lessons are more productive. The findings obtained from this study also support the studies in the literature. The negative aspects of having STEM activities done in the course are determined as time and material shortage, lack of economy, teacher's lack of knowledge and student's getting bored and disconnected from the course. Introduction Teachers have a critical importance in carrying out STEM education in accordance with its purpose. Therefore, it is necessary to reveal the thoughts of teachers about STEM education and to determine its applicability in the classroom environment (Eroğlu & Bektaş, 2016). It is thought that the research can overcome the deficiencies of teachers in this area and will help teachers who will start a master's degree in science education (Eroğlu & Bektaş, 2016). When the literature is EURASIA JOURNAL OF TEACHER EDUCATION, 2021, 2(3), 221-237 VIEWS IN SCIENCE EDUCATION ON STEM EDUCATION 236 examined, there are many studies on taking the opinions of teachers on STEM education (Bakırcı & Kutlu, 2018; Güldemir & Çınar, 2017; Kaya, Şahin & Uz-Özkılıç, 2020; Sözen & Alagöz, 2021). However, studies in which science education graduate students' views on STEM education are determined are mostly in the form of examining existing studies (Elmalı & Balkan-Kıyıcı, 2017; Ceylan, 2021; Herdem & Ünal, 2018). However, science graduate students who have received sufficient STEM education or have applied it in their classroom can contribute positively to the literature on this subject. Therefore, the aim of the research is to determine the views of science teacher graduate students about STEM education. In line with this purpose, “What are the determination of master's students’ views in science education on STEM education. An answer to the problem situation has been sought. Method In the research, it is aimed to determination of master's students’ views in science education on STEM education. For this purpose, the case study method, one of the qualitative research methods, was used in the study. A case study is a qualitative research approach in which the researcher examines the limited situations in a certain time period and defines the situations and the themes related to the situation (Creswell, 2003). The most distinctive feature of the case study is that it allows a detailed examination of the subject. In other words, it reveals the subject covered with a holistic approach. The situation examined in the research is the views of science teacher graduate students about STEM education. The study group of the research consists of 18 science education graduate students who are taking courses at Bayburt University Graduate Education Institute in the fall semester of the 2021-2022 academic year. Participants consist of 11 women and 7 men. 9 of the female participants and 6 of the male participants are teaching science in various institutions. Purposive sampling method, which is one of the non-random sampling methods, was used while determining the participants. In this type of sample, besides the features such as time, money, place and location, the applicability of the subject is also important (Merriam, 2013; Yıldırım & Selvi, 2018). Results Conclusion and Discussion Considering the opinions of the science graduate students participating in the research about STEM education, it was seen that it was necessary to provide teachers with sufficient level of STEM education. Therefore, it can be suggested that education faculties should innovate in their curricula so that they can provide STEM education at an adequate level. Because the practitioners of STEM activities are teachers, and teachers who do not have enough field knowledge cannot teach lessons efficiently. It can be suggested that institutes providing graduate education should give lectures on STEM education. Master's students who are in the thesis writing period should be encouraged to work on STEM education. In this way, more contributions can be made to the literature on STEM education. In addition, it may be recommended that teachers make their students scan the current literature during their graduate education. It can be recommended to GÜRBÜZ ET AL. increase the number of STEM-based course activities in the classroom. In this way, the level of understanding of the science course, which includes a lot of abstract concepts, can be increased. It can be suggested that teachers who want to apply STEM activities in their classes give importance to process evaluation and increase the use of materials. Considering the opinions of the participants who stated that they could not find enough time to implement the STEM activity, it is seen that the current science curriculum should be revised and redesigned in an integrated manner with STEM applications. This study was carried out with 18 teachers consisting of science education graduate students in the fall semester of 2021-2022. Studies can be conducted with different sample groups in which opinions about STEM education will be examined. EURASIA JOURNAL OF TEACHER EDUCATION, 2021, 2(3), 221-237 EURASIA JOURNAL OF TEACHER EDUCATION, 2021, 2(3), 221-237 237 GÜRBÜZ ET AL.
https://openalex.org/W3131598995	https://www.frontiersin.org/articles/10.3389/fmicb.2021.518865/pdf	English	null	Metagenomic Analysis Reveals Microbial Community Structure and Metabolic Potential for Nitrogen Acquisition in the Oligotrophic Surface Water of the Indian Ocean	Frontiers in microbiology	2,021	cc-by	11,542	Edited by: Gipsi Lima Mendez, Catholic University of Louvain, Belgium Edited by: Gipsi Lima Mendez, Catholic University of Louvain, Belgium Edited by: Gipsi Lima Mendez, Catholic University of Louvain, Belgium 1 BGI-Shenzhen, Shenzhen, China, 2 Department of Biotechnology and Biomedicine, Technical University of Denmark, Kongens Lyngby, Denmark, 3 BGI Education Center, University of Chinese Academy of Sciences, Beijing, China, 4 State Key Laboratory of Marine Environmental Science, College of the Environment and Ecology, Xiamen University, Xiamen, China, 5 Third Institute of Oceanography, Ministry of Natural Resources, Xiamen, China, 6 BGI-Qingdao, BGI-Shenzhen, Qingdao, China, 7 State Key Laboratory of Agricultural Genomics, BGI-Shenzhen, Shenzhen, China Reviewed by: Ahmed A. Shibl, New York University Abu Dhabi, United Arab Emirates Isabel Ferrera, Instituto de Ciencias del Mar, Consejo Superior de Investigaciones Cientíﬁcas (CSIC), Spain Reviewed by: Ahmed A. Shibl, New York University Abu Dhabi, United Arab Emirates Isabel Ferrera, Instituto de Ciencias del Mar, Consejo Superior de Investigaciones Cientíﬁcas (CSIC), Spain Despite being the world’s third largest ocean, the Indian Ocean is one of the least studied and understood with respect to microbial diversity as well as biogeochemical and ecological functions. In this study, we investigated the microbial community and its metabolic potential for nitrogen (N) acquisition in the oligotrophic surface waters of the Indian Ocean using a metagenomic approach. Proteobacteria and Cyanobacteria dominated the microbial community with an average 37.85 and 23.56% of relative abundance, respectively, followed by Bacteroidetes (3.73%), Actinobacteria (1.69%), Firmicutes (0.76%), Verrucomicrobia (0.36%), and Planctomycetes (0.31%). Overall, only 24.3% of functional genes were common among all sampling stations indicating a high level of gene diversity. However, the presence of 82.6% common KEGG Orthology (KOs) in all samples showed high functional redundancy across the Indian Ocean. Temperature, phosphate, silicate and pH were important environmental factors regulating the microbial distribution in the Indian Ocean. The cyanobacterial genus Prochlorococcus was abundant with an average 17.4% of relative abundance in the surface waters, and while 54 Prochlorococcus genomes were detected, 53 were grouped mainly within HLII clade. In total, 179 of 234 Prochlorococcus sequences extracted from the global ocean dataset were clustered into HL clades and exhibited less divergence, but 55 sequences of LL clades presented more divergence exhibiting different branch length. Metagenomic Analysis Reveals Microbial Community Structure and Metabolic Potential for Nitrogen Acquisition in the Oligotrophic Surface Water of the Indian Ocean Yayu Wang1,2†, Shuilin Liao1,3†, Yingbao Gai4,5, Guilin Liu6, Tao Jin6, Huan Liu1,7, Lone Gram2, Mikael Lenz Strube2, Guangyi Fan6, Sunil Kumar Sahu1,7, Shanshan Liu6, Shuheng Gan1, Zhangxian Xie4, Lingfen Kong4, Pengfan Zhang1, Xin Liu1,7* and Da-Zhi Wang4* The genes encoding enzymes related to ammonia metabolism, such as urease, glutamate dehydrogenase, ammonia transporter, and nitrilase presented higher abundances than the genes involved in inorganic N assimilation in both microbial community and metagenomic Prochlorococcus population. Furthermore, genes associated with dissimilatory nitrate reduction, denitriﬁcation, nitrogen ﬁxation, nitriﬁcation and anammox were absent in metagenome Prochlorococcus population, Correspondence: Xin Liu liuxin@genomics.cn Da-Zhi Wang dzwang@xmu.edu.cn †These authors have contributed equally to this work Correspondence: Xin Liu liuxin@genomics.cn Da-Zhi Wang dzwang@xmu.edu.cn †These authors have contributed equally to this work Specialty section: This article was submitted to Aquatic Microbiology, a section of the journal Frontiers in Microbiology Received: 10 December 2019 Accepted: 25 January 2021 Published: 18 February 2021 Citation: Wang Y, Liao S, Gai Y, Liu G, Jin T, Liu H, Gram L, Strube ML, Fan G, Sahu SK, Liu S, Gan S, Xie Z, Kong L, Zhang P, Liu X and Wang D-Z (2021) Metagenomic Analysis Reveals Microbial Community Structure and Metabolic Potential for Nitrogen Acquisition in the Oligotrophic Surface Water of the Indian Ocean. Front. Microbiol. 12:518865. doi: 10.3389/fmicb.2021.518865 Specialty section: This article was submitted to Aquatic Microbiology, a section of the journal Frontiers in Microbiology Received: 10 December 2019 Accepted: 25 January 2021 Published: 18 February 2021 Citation: Wang Y, Liao S, Gai Y, Liu G, Jin T, Liu H, Gram L, Strube ML, Fan G, Sahu SK, Liu S, Gan S, Xie Z, Kong L, Zhang P, Liu X and Wang D-Z (2021) Metagenomic Analysis Reveals Microbial Community Structure Specialty section: This article was submitted to Aquatic Microbiology, a section of the journal Frontiers in Microbiology Received: 10 December 2019 Accepted: 25 January 2021 Published: 18 February 2021 Specialty section: This article was submitted to Aquatic Microbiology, a section of the journal Frontiers in Microbiology Received: 10 December 2019 Accepted: 25 January 2021 Published: 18 February 2021 ORIGINAL RESEARCH published: 18 February 2021 doi: 10.3389/fmicb.2021.518865 INTRODUCTION nitrate, nitrite and ammonia, and these nutrients are suﬃcient to support microbial growth in coastal and upwelling areas (Li Y.-Y. et al., 2018; Pajares and Ramos, 2019). However, these N nutrients are extremely low in quantity throughout much of the surface oligotrophic ocean (Moore et al., 2013) and cannot support microbial growth. Instead, microbes have evolved diverse adaptive strategies to ambient N deﬁciency, for example, utilizing small but rapidly cycling dissolved organic nitrogen (DON), such as free amino acids, amines and urea as N source for cell growth and proliferation, indicating the essential roles of DON in maintaining microbial communities in the oligotrophic ocean (Wheeler and Kirchman, 1986; Zubkov et al., 2003; García- Fernández et al., 2004). However, little is known concerning nitrogen acquisition by microbes in the oligotrophic Indian Ocean (Kumar et al., 2009; Díez et al., 2016; Qian et al., 2018; Baer et al., 2019), which impedes our understanding of the mechanisms underlying the adaptive strategies of microbes to ambient N deﬁciency. The Indian Ocean is one of the largest oligotrophic water bodies, which covers approximately one-ﬁfth of global ocean (Eakins and Sharman, 2010; Wei et al., 2019). As the warmest ocean on the earth, the Indian Ocean possesses unique biophysical properties that strongly inﬂuence the diversity and performance of its biota (Massana et al., 2011; Williamson et al., 2012; Díez et al., 2016; Thompson et al., 2017). However, the Indian Ocean is one of the least studied oceans regarding microbial community structure, functional capacity and the potential linkage of microbial taxa and environmental conditions, when compared with other oceans (Venter et al., 2004; Joint et al., 2011; Sjöstedt et al., 2014; Mende et al., 2017; Li Y. et al., 2018). A recent study on biodiversity and spatial distribution of bacteria in the water column of the eastern Indian Ocean has shown that Cyanobacteria and Actinobacteria are more predominant in the upper ocean while Alphaproteobacteria occur more frequently in the deeper layer (Wang et al., 2016). However, a study in the South Indian Ocean has indicated that Gammaproteobacteria dominate the microbial community and their potential functionality is shaped by the depth-related environmental parameters of the Agulhas Current (Phoma et al., 2018). Metagenomic analysis of the picocyanobacterial community in the equatorial waters of the Indian Ocean reveals that the genera Prochlorococcus and Synechococcus comprise 90% of the cyanobacterial reads (Díez et al., 2016). Citation: Wang Y, Liao S, Gai Y, Liu G, Jin T, Liu H, Gram L, Strube ML, Fan G, Sahu SK, Liu S, Gan S, Xie Z, Kong L, Zhang P, Liu X and Wang D-Z (2021) Metagenomic Analysis Reveals Microbial Community Structure and Metabolic Potential for Nitrogen Acquisition in the Oligotrophic Surface Water of the Indian Ocean. Front. Microbiol. 12:518865. doi: 10.3389/fmicb.2021.518865 February 2021 \| Volume 12 \| Article 518865 Frontiers in Microbiology \| www.frontiersin.org 1 Wang et al. Microbial Community of Indian Ocean i.e., nitrogenase and nitrate reductase. Notably, the de novo biosynthesis pathways of six different amino acids were incomplete in the metagenomic Prochlorococcus population and Prochlorococcus genomes, suggesting compensatory uptake of these amino acids from the environment. These results reveal the features of the taxonomic and functional structure of the Indian Ocean microbiome and their adaptive strategies to ambient N deﬁciency in the oligotrophic ocean. Keywords: microbe, Prochlorococcus, metagenome, nitrogen metabolism, Indian Ocean INTRODUCTION Prochlorococcus populations in the ocean have been deﬁned with high light-adapted (hereafter HL) populations and low light-adapted (hereafter LL) populations, where Prochlorococcus HLIIA ecotype is abundant in the Prochlorococcus community in the Indian Ocean (Farrant et al., 2016). All together, these studies have provided a snapshot of the microbial diversity and geographical distribution in some speciﬁc areas of the Indian Ocean, but knowledge on the overall microbial taxonomic structure and functional capacity, and their inﬂuencing factors are still very limited. Metagenomic studies of the global ocean advance our understanding of diversity, evolution and functional potential of natural microbial communities (Sunagawa et al., 2015; Li Y. et al., 2018). Distribution of microbial diversity and biogeochemistry are structured largely by environmental gradients such as light, temperature, oxygen, salinity, and nutrients (Thompson et al., 2017). In this study, we applied a metagenomic approach to investigate microbial communities in the oligotrophic surface waters of the Indian Ocean, from the Andaman Sea in the east to the Red Sea in the west, and characterized their nitrogen acquisition strategies based on a 9.5 million (M) microbial gene set (Figure 1 and Supplementary Figure S1). We paid particular attention to the predominant cyanobacterial genus Prochlorococcus and their nitrogen assimilation strategies due to its signiﬁcant contribution to the stability, resilience and function of the marine ecosystem. This study expands our understanding of the microbial community and their metabolic potentials in the Indian Ocean, and provides fundamental metagenomic data for further microbial studies in the Indian Ocean, which is a signiﬁcantly understudied realm. Nitrogen (N) is an essential macronutrient limiting microbial growth and proliferation in the ocean, especially in the oligotrophic oceanic regimes (Zehr and Wad, 2002; Moore et al., 2013). Ambient N deﬁciency aﬀects N assimilation, carbon ﬁxation, photosynthesis, pigment and lipid accumulation of microbes (Schwarz and Forchhammer, 2005). Typically, a broad diversity of prokaryotes from Proteobacteria, Firmicutes, Verrucomicrobia, Planctomycetes, Acidobacteria, Chloroﬂexi, and Chlorobia can use dissolved inorganic N (DIN), such as Frontiers in Microbiology \| www.frontiersin.org MATERIALS AND METHODS Sampling and DNA Extraction A total of 17 surface seawater samples (S6–S38, 5 m depth) were collected from the outlet of proﬁling CTD (Seabird SBE21) during the 26th cruise of China Ocean Mineral Resources R Sampling and DNA Extraction g A total of 17 surface seawater samples (S6–S38, 5 m depth) were collected from the outlet of proﬁling CTD (Seabird SBE21) during the 26th cruise of China Ocean Mineral Resources R February 2021 \| Volume 12 \| Article 518865 Frontiers in Microbiology \| www.frontiersin.org 2 Wang et al. Microbial Community of Indian Ocean FIGURE 1 \| Sampling locations and the microbial genes found in the Indian Ocean. (A) Sampling stations across the Indian Ocean and Red Sea. (B) The number of shared genes (orange) between the 17 SE sample gene set (gray) and three PE sample assembled gene set (green). 1.3 M and 1.7 M genes from two gene sets were shared, which occupied 17.06 and 48.3% of the gene numbers in two gene sets, respectively. FIGURE 1 \| Sampling locations and the microbial genes found in the Indian Ocean. (A) Sampling stations across the Indian Ocean and Red Sea. (B) The number of shared genes (orange) between the 17 SE sample gene set (gray) and three PE sample assembled gene set (green). 1.3 M and 1.7 M genes from two gene sets were shared, which occupied 17.06 and 48.3% of the gene numbers in two gene sets, respectively. & D Association (COMRA1) on the R/V DayangYihao in May 2012. The sampling stations ranged across the Indian Ocean, from the Andaman Sea in the east to the Red Sea in the west (Figure 1A). Samples from the Red Sea were speciﬁcally referred to as S32, S34, S36, and S38. For each sample, more than 200 l of seawater was preﬁltered using a GF/A glass ﬁber membrane (1.6 µm, 142 mm ﬁlter diameter, Whatman) with a Flojet Pump (04300242A) and then collected on a Supor polyethersulfone membrane (0.2 µm, 142 mm ﬁlter diameter, Pall). The membranes were placed in 50 ml tubes and immediately frozen in liquid nitrogen, and then stored at −80◦C until further analysis. DNA extraction was performed using the PowerSoil DNA isolation kit (QIAGEN, United States) according to the instructions. Environmental parameters were also monitored, including temperature, salinity, pH and inorganic nutrients listed in Supplementary Table S1. Concentrations of phosphate, silicate, ammonium, nitrite and nitrate as well as chlorophyll-a concentration were measured in the laboratory according to the national standard protocol for investigation of chemical elements of seawater titled “Speciﬁcations for oceanographic survey-Part 4: Survey of chemical parameters in seawater” (GB/T 12763.4-2007, 2007). Construction of the Gene Catalog Construction of the Gene Catalog After a quality check and pre-processing by SOAP nuke (v1.5.3) (Chen et al., 2017), all clean reads of BGISEQ were aligned against the Ocean Microbiome Reference Gene Catalog (OM-RGC) (Sunagawa et al., 2013) using bowtie2.2.6 (Langmead and Salzberg, 2012) with parameters: bowtie2 –q – phred33 –sensitive –mixed –no-discordant -p 20 -k 200 -x. Then microbial genes mapped by unique reads were considered to be present in the samples. On average, 72.68% of BGISEQ SE reads were mapped to the OM-RGC, resulting in an average of 2.92 M genes in each sample (Supplementary Table S2). All the genes identiﬁed from 17 samples were merged together and then duplicates were removed, generating a 7.7 M unique gene set. We named this set the 17 SE sample gene set (Supplementary Table S2). 1http://www.comra.org/ Sampling and DNA Extraction sequencing on the BGISEQ-500 sequencer with the SE50 mode. The sequence images were base-called by the software Zebra call (base calling software developed for BGISEQ-500). A total of 12.81 billion raw reads were produced by the BGISEQ-500 platform, with a read length of 50 bp. Since long and pair-end (PE) reads are more useful for metagenome assembly, three representative local samples were collected from the stations of S8 (E 93◦49.228′, N 5◦54.323′), S18 (E 72◦52.169′, N 1◦42.848′) and S26 (E 56◦5.870′, N 13◦26.297′) located at the east, central and west regions of the Indian Ocean, and were selected for in-depth sequencing using a 150 bp PE strategy on Hiseq4000 platform (Illumina). A total of 749.03 M reads were generated with an average 249.6 M reads for each sample. DNA Sequencing on BGISEQ-500 and HiSeq4000 All 17 DNA samples were sequenced using a 50 bp single-ended (SE) sequencing strategy on a BGISEQ500 platform (Beijing Genomics Institute, Shenzhen). Firstly, the DNA sample was fragmented by ultrasound on Covaris E220 (Covaris, Brighton, United Kingdom). Then 400–700 bp DNA fragments were recovered to construct a library for each sample according to standard instructions (Fang et al., 2017). Three barcoded libraries were pooled together with equal amounts to make DNA nanoballs (DNB). Each DNB was loaded into one lane for After quality control, the Illumina PE reads of three representative local samples (S8, S18, and S26) were de novo assembled into contigs using MegaHit (v1.0) with parameters: - t 20 -min-contig-len 500 –presets meta-large –min-count 5 (Li et al., 2016). A total of 23.04% of the Illumina PE reads were assembled into 3.26 M contigs (= 500bp). GeneMarkS (version February 2021 \| Volume 12 \| Article 518865 Frontiers in Microbiology \| www.frontiersin.org 3 Microbial Community of Indian Ocean Wang et al. 2.7) (Besemer et al., 2001) was used to predict the genes using the parameter: gmhmmp -m MetaGeneMark_v1.mod in contigs obtained from each sample. The length of the predicted genes was no less than 150 bp. A non-redundant gene catalog (3.4M) was built by clustering all predicted genes using CDHIT (version 4.6.5) (Li and Godzik, 2006) with the following parameters: 95% identity and 90% coverage. The 3.4 M assembled gene set (called three PE sample gene set) was then compared with the 7.7 M gene set (17 SE sample gene set) to identify the overlapping genes using CDHIT with the same cutoﬀas above. As the shared genes accounted for a low proportion (Figure 1B) of the two gene sets, we merged these two gene sets together. We then removed the redundancy to generate a more complete gene set for the Indian ocean samples. to the Akaike information criterion (AIC). The correlations of the relative abundance of Prochlorococcus with nitrate, nitrite and ammonium concentrations were separately calculated using Spearman’s rank correlation test using the function cor. test () in R package (version 3.5.3). Phylogenetic Tree of Prochlorococcus Genomes A total of 201 Prochlorococcus genomes including 27 known ecotype genomes (Biller et al., 2014) were downloaded from NCBI (Supplementary Table S3). The gene prediction of each genome was performed using GeneMarkS (version 2.7) (Besemer et al., 2001) with the parameter: gmhmmp -m Prochlorococcus_preﬁx.mod. All genes were merged together to construct a pangenome. Then the SE reads from each sample were mapped against the pangenome using bowtie2.2.6 to identify Prochlorococcus genes. If the portion of gene numbers mapped by reads was more than 50%, the Prochlorococcus strain was considered to be present in the sample. The abundance of each identiﬁed gene was calculated by dividing the mapping reads with the gene length. The abundance of the identiﬁed Prochlorococcus was represented by the average abundance of their identiﬁed genes. The same 40 single copy genes (SCGs) (Mende et al., 2013) from the 55 identiﬁed strains and 25 known ecotype strains were predicted by fetchMG (Kultima et al., 2012; Sunagawa et al., 2013). Then multiple alignments of 40 SCGs were performed using Maﬀt (Kazutaka and Daron, 2013). Here we selected 25 of 27 known ecotype strains as reference because we could not extract a full number of SCGs genes from the remaining two strains genomes. A phylogenic tree was constructed using ETE3 and the standard FastTree workﬂow (Price et al., 2010; Huerta-Cepas et al., 2016a). Taxonomic and Functional Annotation Taxonomic and Functional Annotation Taxonomic and functional assignment of genes of the complete gene set was performed. Taxonomic assignments were performed by aligning protein sequence to the NCBI-NR database using DIAMOND (version 0.8.22) (Buchﬁnk et al., 2015). The top alignment hits were retained according to the criteria of coverage ≥80%, identity ≥65%, and e-value ≤1e- 5 as previous studies described (Qin et al., 2010; Li et al., 2014). Then, the taxonomic annotation of each gene was determined by the lowest common ancestor (LCA)-based algorithm implemented in MEGAN (Huson et al., 2007). Functional annotations were made by aligning the protein sequence against eggNOG (version 4.5) (Huerta-Cepas et al., 2016b) and KEGG (version.81) database by the highest scoring annotated hit(s) containing at least one high-scoring pair (HSP) scoring over 60 bits by DIAMOND. Frontiers in Microbiology \| www.frontiersin.org Analysis of N Metabolism and Amino Acids Biosynthesis The KOs involved in N metabolism were extracted from the merged gene set to analyze functional capacity of the Indian Ocean microbiome. The KOs involved in N metabolism and the annotated Prochlorococcus data were extracted to explore the N assimilatory pathways of the Prochlorococcus population in the Indian Ocean. The key enzymes were also manually annotated using the Pfam database to validate the existence of functional domain. All KOs in map01230-Biosynthesis of amino acids were extracted to explore biosynthesis reactions of amino acids. The KOs functionally annotated with “transporter” were extracted from the metagenome of Prochlorococcus population and the identiﬁed single Prochlorococcus genome respectively to predict the uptake model of amino acids from the environment. The single Prochlorococcus genome was identiﬁed based on 201 published Prochlorococcus genomes and the Indian Ocean metagenome sequencing data, and the detailed process was described below. Microbial Gene Capacity in the Indian Ocean The locations of the 17 sampling stations along an east-west transect in the Indian Ocean (12◦S, 96◦E to 4◦S, 39◦E) are shown in Figure 1A. In total, 12.68 billion high quality SE reads were generated, resulting in an average of 37.16 Gb data per sample. Here, the microbial functional genes were identiﬁed by mapping these SE reads to the Tara Oceans gene set (OM-RGC, 40M genes). An average of 3 M genes per sample were identiﬁed with a 72.68% mapping rate. The reads of three PE sequenced samples were also mapped to OM-RGC and 94.90% of identiﬁed genes by PE reads were covered by SE reads (Supplementary Figure S2), indicating that the SE data was suﬃcient to map reference genes in this study. By merging all identiﬁed microbial genes of the 17 SE sequenced samples together, a 17 SE sample gene set was generated, comprising 7.7 M non-redundant genes (Supplementary Table S2). y To mine more speciﬁc microbial genes in the Indian Ocean surface water, a 3.4 M gene set based on de novo assembly was generated using three PE sequenced samples, which obtained 51.95–52.16% mapping rate in individual samples. The shared genes between the 17 SE sample gene set and three PE sample assembled gene set accounted for an average of 17.06 and 43.82% of gene abundance in the SE mapping gene set and PE assembly gene set (Figure 1B), suggesting that many novel genes could be explored by de novo assembly from local samples. So, a new complete microbial gene set (9.5M) from the Indian Ocean surface waters was generated by the combination of OM-RGC mapping and de novo assembly methods. Based on the new gene set, many more genes could be identiﬁed with higher reads mapping rate in each sample. The clustering analysis based on physicochemical properties of all samples showed that the 17 samples were divided into three groups, the three Red Sea samples (S34, S36, and S38) were grouped together with the sample S30 from the west of the Indian Ocean, the Red Sea sample S32 was clustered with the samples S18, S22, S24, S26, which were located in the middle of the Indian Ocean, while the other remaining samples were grouped together (Supplementary Figure S4). These results demonstrated that the physicochemical properties of the west region of the Red sea was distinguishable from the Indian Ocean. Statistical Analyses The relative abundances of genes were calculated based on the unique mapping reads as previously described (Qin et al., 2012). Firstly, the read counts from each gene were normalized by dividing with the gene length to calculate gene abundance. Then the relative abundance of each gene in samples were generated by dividing with the sum of abundance of all genes. Each KO abundance was generated by summing up the relative abundance of genes annotated to the same KO. The taxonomic proﬁle was constructed using the same method. The meta- genes (unigenes) in the metagenomic gene set assigned to the genus of Prochlorococcus were extracted and the sum of their abundance in each sample was determined as the abundance of Prochlorococcus. The diﬀerence in abundance of Prochlorococcus between the central ocean and the coast samples, and the diﬀerences in dissolved inorganic nitrogen (DIN) concentration between the central ocean samples and coast samples were checked using t-test (basic function in R version 3.5.3). The clustering analysis based on physicochemical properties of all samples was performed using function pheatmap () in R package (version 3.5.3). To determine the shaping factors causing the variation in microbial communities among samples, the environmental variables were included in a redundancy analysis (RDA) model using the function rda () in “vegan” R-package, which was then reduced by the step-function in R according To evaluate the diversity of Prochlorococcus population in the global ocean, 40 SCGs were extracted from 201 reference genomes, OM-RGC and the Indian Ocean gene set by fetchMG. February 2021 \| Volume 12 \| Article 518865 4 Microbial Community of Indian Ocean Wang et al. genera in the Bacteroidetes phylum presented similar relative abundances, such as the dominant genera Fluviicola (1.53% of relative abundance) and Flavobacterium (0.97% of relative abundance). The Actinobacteria were represented mainly by Streptomyces (5.41% of relative abundance) and Candidatus actinomarina (4.81% of relative abundance). The Firmicutes were mainly composed of Clostridium (8.80% of relative abundance) and Bacillus (8.83% of relative abundance). The genera Coraliomargarita (25.89% of relative abundance), Pedosphaera (11.05% of relative abundance) and Verrucomicrobium (5.24% of relative abundance) dominated the phylum of Verrucomicrobia. While Rhodopirellula (25.94% of relative abundance) and Planctomyces (12.13% of relative abundance) were the abundant groups of Planctomycetes. Among them, the photosynthetic cyanobacterial taxa such as Prochlorococcus and Synechococcus were the most abundant in each sample, comprising 86.11% of total cyanobacterial sequences (Figure 2A and Supplementary Table S4). Microbial Gene Capacity in the Indian Ocean RDA was performed to discern the relationship between microbial community structure and environmental parameters such as concentrations of phosphate, silicate, ammonium, nitrite, nitrate, as well as temperature, salinity and pH (Supplementary Table S1). A model describing the environmental parameters that were signiﬁcantly correlated with microbial composition was selected based on Akaike information criterion, indicating that temperature, phosphate, silicate and pH rather than ammonium, nitrite, nitrate and salinity had signiﬁcant eﬀect on the microbial composition. The two axes could explain 40.5% of the overall variance in community structure with an adjusted R2 (0.56), showing a remarkable correlation. The Statistical Analyses Particularly, Synechococcus with higher relative abundance (average 25.9% of total bacterial sequence) predominated in the samples collected from S7, S8, and S32 stations close to the coastal areas with relatively high DIN concentration while Prochlorococcus dominated with an average 17.4% relative abundance in the middle area with low DIN concentration (Supplementary Table S1, t-test, P-value < 0.05) (Figure 2A). Besides, no obvious diﬀerence in microbial composition between the Red Sed and other locations of the Indian Ocean. Notably, the abundance of Prochlorococcus was negatively correlated with the abundance of Synechococcus across the transect (P-value = 2.2e−16, Spearman’s rank correlation test with the correlation coeﬃcient = −0.88). Although the DIN concentrations of the Indian Ocean surface waters varied among stations, it was much lower than the minimum detection limit and the N/P ratio was less than 8 (Supplementary Table S1), indicating that the Indian Ocean surface water belonged to an oligotrophic water body. Among all the screened genes, the gene family COG0172 (Seryl- tRNA synthetase) showed the highest number, therefore, it was selected to construct the phylogenetic tree. Only the genes having a sequence length higher than 800 bp were retained in the tree. Taxonomic and Functional Variations in the Indian Ocean A large proportion of genes (51.91%) in the complete gene set belong to bacteria, while 7.96%, 2.00% and 1.10% were annotated to virus, eukaryote and archaea, respectively (Supplementary Figure S3A). More than 69.51% of bacterial sequences could be annotated at the phylum level. Proteobacteria and Cyanobacteria dominated the microbial community by occupying an average 37.85 and 23.56% of total bacterial sequences of the Indian Ocean, respectively, followed by Bacteroidetes (3.73%), Actinobacteria (1.69%), Firmicutes (0.76%), Verrucomicrobia (0.36%), and Planctomycetes (0.31%) (Supplementary Figure S3B). Totally, 684 bacterial genera were found in the Proteobacteria phylum, of which the genus Candidatus pelagibacter was dominant with an average of 12.03% of relative abundance (Supplementary Table S4). The February 2021 \| Volume 12 \| Article 518865 Frontiers in Microbiology \| www.frontiersin.org 5 Microbial Community of Indian Ocean Wang et al. FIGURE 2 \| Microbial communities in the surface waters of the Indian Ocean. (A) Microbial community structure with abundant Prochlorococcus and Synechococcus. The samples from stations S7, S8, and S32 near the coast with distinct taxonomic compositions distinct from the other samples. (B) Redundancy analysis of bacterial community structure and environmental variables. (C) Distributions of typical metabolism pathways among different sampling stations. FIGURE 2 \| Microbial communities in the surface waters of the Indian Ocean. (A) Microbial community structure with abundant Prochlorococcus and Synechococcus. The samples from stations S7, S8, and S32 near the coast with distinct taxonomic compositions distinct from the other samples. (B) Redundancy analysis of bacterial community structure and environmental variables. (C) Distributions of typical metabolism pathways among different sampling stations. parameters of temperature, phosphate, silicate and pH drove the composition variance and separated samples into diﬀerent groups, of which temperature was the most important and could explain the most variation in response variables (Figure 2B). It was found that the three western Red sea samples were grouped in the upper right of the RDA plot, and were mainly associated with concentrations of phosphate and silicate. But most of the Indian Ocean samples (except samples S7 and S8 near the coast) were in the lower left and were associated with temperature and pH. These results indicated that the variation among microbial communities in the Red Sea and in the Indian Ocean were driven by diﬀerent environmental factors. In addition, the dominant genus Prochlorococcus was positively associated with temperature and pH in the multivariate analysis, while the genus Synechococcus was negatively associated with pH. Taxonomic and Functional Variations in the Indian Ocean Interestingly, the concentrations of ammonium, nitrate and nitrite were not associated with the variation of the whole microbial community. However, the abundance of Prochlorococcus presented a remarkably negative correlation with nitrite concentration (P-value < 0.01, Spearman’s rank correlation test), but not with the concentrations of ammonium, nitrate, phosphate and other factors, indicating that nitrite impacted Prochlorococcus although it had no eﬀect on the whole microbial composition of the Indian Ocean. metabolic function (carbohydrates, amino acids, nucleotide, cofactors and vitamins, lipid, energy metabolism), genetic information processing (replication and repair), secondary metabolite biosynthesis (glycan, terpenoids, and polyketides), and signal transduction, and the majority of them corresponded to housekeeping functions. The abundances of 15 typical pathways were calculated using a total abundance of marker KOs divided by the number of KOs as described in a previous study (Sunagawa et al., 2015). All samples showed a similar variation pattern in functional composition (Figure 2C). Function modules associated with iron-related metabolism, carbon ﬁxation (photosynthesis), carbon ﬁxation (Prok), oxygenic photosynthesis, sulfur and phosphorus metabolism with high abundances co-existed in all samples. In addition, other processes such as nitrogen metabolism, methanogenesis, manganese–related metabolism, ﬂagellar assembly, bacterial chemotaxis, bioﬁlm formation, aerobic respiration and anoxygenic photosynthesis with low abundances were prevalent in each sample. Frontiers in Microbiology \| www.frontiersin.org Prochlorococcus Genomes in the Indian Ocean and the Global Ocean Four metabolic reactions involved in nitrogen metabolism including dissimilatory nitrate reduction, assimilatory nitrate reductase, denitriﬁcation and nitriﬁcation were detected in our metagenomic dataset (Figure 4C). Although the assimilatory nitrate reduction (narB, nasA, narsB, and nirA) presented high abundance in nitrogen metabolism pathway, it contributed only a small portion to ammonia production. The functional genes related to nitrogen ﬁxation and anammox were not detected in all samples (Figure 4). To characterize nitrogen acquisition of the Prochlorococcus population in the Indian Ocean, all genes involved in nitrogen metabolism from Prochlorococcus genus were extracted from the metagenome. Prochlorococcus could potentially assimilate nitrogen for growth through six diﬀerent routes (Figure 4). Three dominant routes were comprised of hydrolyzing urea by ureas, hydrolyzing nitrile to ammonia by nitrilase and converting glutamine into ammonia by gdhA. The fourth route was to directly transport extracellular ammonia into intracellular via transporter. The ﬁfth route was to uptake extracellular nitrite and nitrate via transporters, and then catalyze the reduction of nitrite to ammonia by NirA and ﬁnally be converted into amino acids. The gene encoding nitrate reductase (NarB) involved in the reduction of nitrate to nitrite was not identiﬁed in the Prochlorococcus population. The nitrite could also be converted by oxidation of nitroalkanes using nitronate monooxygenase. The sixth route was to convert cyanate into ammonia by cyanate lyase. Only the metabolic reaction related to assimilatory nitrate reduction was detected in metagenomic Prochlorococcus population. The other key enzymes related to dissimilatory nitrate reduction, denitriﬁcation, nitrogen ﬁxation, nitriﬁcation and anammox were not found (Figure 4C). Collectively, the Prochlorococcus population in the Indian Ocean could assimilate urea, ammonia, nitrite, nitroalkane, cyanate, and nitrile as nitrogen sources, but they lost the ability to assimilate nitrate and nitrogen due to the lack of NarB and nifH. To further characterize the diversity of Prochlorococcus in the global ocean, the 40 SCGs of Prochlorococcus genus were fetched from the Tara Ocean gene set, the PE sample assembled gene set and 201 Prochlorococcus strains. As the multiple sequence alignment was not suitable for the SCGs from metagenome data, a phylogenetic tree of Prochlorococcus was constructed based on one gene, namely COG0172 (Seryl-tRNA synthetase) as its highest number in diﬀerent data sources. This gene is an evolutionarily conserved enzyme which catalyzes the formation of aminoacyl-tRNAs that is used as substrates for ribosomal protein biosynthesis. The Prochlorococcus sequences were divided into two main clades, HL and LL (Figure 3B). Prochlorococcus Genomes in the Indian Ocean and the Global Ocean Prochlorococcus an extensively studied photosynthetic bacterial taxon, dominated the microbial community of the Indian Ocean surface water with an average abundance of 14.5% among metagenome sequences (Figure 2A). Using the pangenome of the 201 published Prochlorococcus genomes as a reference (Supplementary Table S3), about 14.08% of reads could be Variations of microbial functional composition were examined among stations. Overall, only 24.3% of genes but 82.6% of KOs were co-existed in all samples, indicating a high level of gene diversity and functional redundancy across the Indian Ocean. These shared KOs mainly contributed to February 2021 \| Volume 12 \| Article 518865 Frontiers in Microbiology \| www.frontiersin.org 6 Microbial Community of Indian Ocean Wang et al. mapped to the Prochlorococcus gene set, which was very close to the abundance identiﬁed in the metagenome data, implying the integrity of the reference data set. Totally, 55 Prochlorococcus strains were detected, including 12 cultured strains and 43 uncultured strains. The detections of 55 strains implied the species diversity of Prochlorococcus in the surface water of the Indian Ocean. Of these, scB245a_518D8, scB241_528J8, MIT9301, MIT9302, GP2 and MIT9201 were most abundant Prochlorococcus strains among the samples (Supplementary Figure S5). Only 5 out of 12 cultured strains are within known HLII ecotype. All of 43 uncultured single-cell ampliﬁed genome and metagenome-assembled genomes have no ecotype information. To explore the evolutional relationship of these Prochlorococcus strains, phylogenetic clades of all these unknown strains (combined with 25 cultured strains with representative ecotypes) were explored using an alternative method based on 40 SCGs. The phylogenetic tree showed that all the identiﬁed genomes were divided into two major known ecotypes: HLI and HLII clades (Figure 3A), in accordance with their niches. Except for the strain UBA3999, which was ﬁnally validated to be aﬃliated to Synechococcus as only 70.81% average nucleotide identity (ANI) to Prochlorococcus strain MIT0701 and 30 conserved SCGs consistently annotated to Synechococcus, 53 of 54 Prochlorococcus strains in the Indian Ocean surface waters belonged to the HLII ecotype and only one strain belonged to the HLI ecotype. The ecotype-level community structure was similar across the Indian Ocean, but the relative abundances of Prochlorococcus strains varied among diﬀerent stations. For example, the composition of Prochlorococcus strains in the Red Sea samples (S30, S32, S34, and S38) were clustered together and were more similar to the west locations of the Indian Ocean (Supplementary Figure S5). formamidase (Figures 4A,B). Prochlorococcus Genomes in the Indian Ocean and the Global Ocean Of these, 179 of 234 sequences were clustered within the HL clade with similar phylogenic distance and 55 of sequences were clustered within LL clades with diﬀerent branch distance. It suggested that the Prochlorococcus sequences in LL clades evolved more divergently, which might be attributed to the speciﬁc features of deep- sea environment. To validate the results from the metagenome, the abilities of nitrogen metabolism from 54 detected Prochlorococcus genomes (including six complete genomes) in the Indian Ocean were evaluated. A similar result was observed in those detected Prochlorococcus genomes that the enzyme nifH involved in nitrogen ﬁxation was absent but the NarB catalyzing nitrate to nitrite was found in 14 strain genomes including two cultured strains (SB, MIT0604) and 12 single-cell ampliﬁed genomes (Supplementary Table S5). These results indicated that most of the Prochlorococcus strains lost the ability to reduce nitrate, but some of them still maintained the ability to assimilate nitrate and could utilize it as N source. Furthermore, the enzymes involved in assimilation of other nitrogen forms such as cyanate, nitrile, nitrite, and amino acids also diﬀered among strains, suggesting their diﬀerent survival strategies. Amino Acid Biosynthesis of Prochlorococcus in the Indian Ocean The relative abundance of ammonia input-related genes such as urease, glutamate dehydrogenase (gdhA) and ammonia transporter (amt) were predominant in each sample, followed by nitrilase, ferredoxin-nitrite reductase (NirA), cyanate lyase and The amino acid biosynthesis model was predicted based on the functional enzymes of amino acid biosynthesis and amino acid transporter proteins in the metagenome of Prochlorococcus February 2021 \| Volume 12 \| Article 518865 Frontiers in Microbiology \| www.frontiersin.org 7 Microbial Community of Indian Ocean Wang et al. FIGURE 3 \| Phylogenetic tree based on single copy genes from the 54 detected strains in the Indian Ocean and Prochlorococcus sequences from the global ocean, respectively. (A) Phylogenetic tree from the detected strains in our data set (names in bold with∗) and representative Prochlorococcus genomes was constructed based on 40 SCGs. Phylogenetic clade afﬁliations are represented by different colors; HLII, light green; HLI, dark green; LLI, median blue; LLII_III, dark blue; LLIV, light blue; Synechococcus, gray; unclassiﬁed strains, yellow. (B) The Phylogenetic tree of Prochlorococcus populations in the global ocean based on gene sequences from COG0172. red, sequences identiﬁed in assembled gene set; gray, sequences from Tara Ocean gene set; yellow, sequences from Prochlorococcus genomes; reference sequences are colored according to their ecotypes. FIGURE 3 \| Phylogenetic tree based on single copy genes from the 54 detected strains in the Indian Ocean and Prochlorococcus sequences from the global ocean, respectively. (A) Phylogenetic tree from the detected strains in our data set (names in bold with∗) and representative Prochlorococcus genomes was constructed based on 40 SCGs. Phylogenetic clade afﬁliations are represented by different colors; HLII, light green; HLI, dark green; LLI, median blue; LLII_III, dark blue; LLIV, light blue; Synechococcus, gray; unclassiﬁed strains, yellow. (B) The Phylogenetic tree of Prochlorococcus populations in the global ocean based on gene sequences from COG0172. red, sequences identiﬁed in assembled gene set; gray, sequences from Tara Ocean gene set; yellow, sequences from Prochlorococcus genomes; reference sequences are colored according to their ecotypes. further validated the reaction gaps in amino acid biosynthesis of the Prochlorococcus population in the Indian Ocean. population. Of the 230 functional KOs involved in amino acid biosynthesis, 96 were found in the Prochlorococcus population, which contributed to de novo biosynthesis of 14 amino acids such as valine, leucine, isoleucine, tryptophan, proline, threonine, cystenine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, and histidine. Amino Acid Biosynthesis of Prochlorococcus in the Indian Ocean However, reaction gaps for the biosynthesis of amino acids existed in six types of amino acids (Table 1). The enzyme metC that synthesizes precursor homocysteine of methionine and the serB that catalyzes phosphoserine to serine were missing. The enzyme glyA that functions bi-directionally transformation of serine and glycine was detected but the bidirection enzyme glyA for glycerine and threonine conversion was absent. The enzymes asdA and ALT for de novo biosynthesis of alanine were also absent. Furthermore, biosynthesis reactions of aromatic amino acids tyrosine and phenylalanine were incomplete due to the lack of the enzyme tyrB that catalyzes the precursor of tyrosine and converts phenylpyruvate into phenylalanine. Interestingly, the functional genes encoding two types of transporter proteins responsible for importing glycine and alanine into cells were found in the metagenome data of the Prochlorococcus population, suggesting that Prochlorococcus absorbed glycine and alanine from the environment. Thus, it could be postulated that serine might be achieved from glycine by glyA, and subsequently, converted to cysteine and methionine. The same observation was also found in the genomes of the 54 Prochlorococcus strains detected, which Frontiers in Microbiology \| www.frontiersin.org DISCUSSION Metagenomic studies of the global ocean advance our understanding of microbial diversity, community structure, functional potential, the environment inﬂuence and biotic interactions (Lima-Mendez et al., 2015; Sunagawa et al., 2015). In this study, we generated a 9.5 M gene set for surface microbes of the Indian Ocean combined with mapped genes from OM-RGC and de novo assembled genes from the local samples. Our results showed that most of the microbial genes of surface waters had been embraced by this gene set and could be captured by sample reads. Although the OM-RGC provides an ecosystem- wide data set, which enables ocean microbial genetic diversity accessible for various targeted studies, it does not cover all microbial functional genes owing to the limitation of samples and sampled regions. Therefore, it would be helpful to combine this data with a small set of assembly genes from local samples. Proteobacteria and Cyanobacteria were the abundant phyla in surface waters of the Indian Ocean, and photosynthetic cyanobacterial taxa such as Prochlorococcus and Synechococcus were dominant in all samples. These results are consistent with the previous studies (Díez et al., 2016; Wang et al., 2016; February 2021 \| Volume 12 \| Article 518865 Frontiers in Microbiology \| www.frontiersin.org Frontiers in Microbiology \| www.frontiersin.org 8 Microbial Community of Indian Ocean Wang et al. GURE 4 \| Metabolic potential for N acquisition in the microbial community and metagenomic Prochlorococcus population of the Indian Ocean. (A) The generic trogen assimilation routes (colored line) in the metagenome and Prochlorococcus population of the Indian Ocean. (B) Abundances of key functional genes involved ammonia production in the metagenome and Prochlorococcus population. (C) The functional models of an N metabolism pathway in the metagenome and ochlorococcus population. FIGURE 4 \| Metabolic potential for N acquisition in the microbial community and metagenomic Prochlorococcus population of the Indian Ocean. (A) The generic nitrogen assimilation routes (colored line) in the metagenome and Prochlorococcus population of the Indian Ocean. (B) Abundances of key functional genes involved in ammonia production in the metagenome and Prochlorococcus population. (C) The functional models of an N metabolism pathway in the metagenome and Prochlorococcus population. FIGURE 4 \| Metabolic potential for N acquisition in the microbial community and metagenomic Prochlorococcus population of the Indian Ocean. (A) The generic nitrogen assimilation routes (colored line) in the metagenome and Prochlorococcus population of the Indian Ocean. Frontiers in Microbiology \| www.frontiersin.org DISCUSSION (B) Abundances of key functional genes involved in ammonia production in the metagenome and Prochlorococcus population. (C) The functional models of an N metabolism pathway in the metagenome and Prochlorococcus population. Phoma et al., 2018). However, variations at the sub-family level classiﬁcation were observed among diﬀerent regions of the Indian Ocean, for example, Synechococcus was abundant in the stations near the coast but Prochlorococcus dominated in the central area of the Indian Ocean, which might be caused by environmental variables. The eﬀects of environmental factors on marine microbial communities have been widely studied (Coutinho et al., 2015). Composition, distribution and metabolic potential of microbes are structured, to a large extent, by environmental gradients in light, temperature, oxygen, salinity and nutrients (Thompson et al., 2017). Globally, temperature is the key environmental factor driving the geographical distribution of microbes in the ocean (Sunagawa et al., 2015). In this study, temperature, phosphate, silicate and pH were important environmental factors regulating microbial distribution in the Indian Ocean. However, the variation of microbial composition in the Red Sea was mainly associated with the concentrations of phosphate and silicate (Figure 2B). It should be noted that the physicochemical properties in the west of the Red Sea diﬀered from the Indian Ocean, resulting in a slight diﬀerence in microbial composition. Investigation of microbial adaptation to environmental changes indicated that temperature explains most of the taxonomic and functional variations in the Red Sea, followed by nitrate, chlorophyll, phosphate, and salinity (Thompson et al., 2017). These highly shared KO functions across the samples might confer a buﬀering capacity for an ecosystem in scenarios of biodiversity loss. The analysis of typical pathways indicated that metabolic processes of microbial community mainly contributed to the production of a diverse array of nutritional substrates such as ferrous/ferric iron, carbohydrate, phosphate, and sulfate. Especially for carbon ﬁxation and oxygenic photosynthesis, they enabled microbes to use organic compounds and molecular oxygen of photosynthetic origin in the oligotrophic oceans. Overall, the microbial gene capacity determines their adaptive ability to environmental variations in the ocean. A comprehensive microbial gene set from various ocean environments would facilitate to fully understand the connection between microbes and environmental variables. February 2021 \| Volume 12 \| Article 518865 Frontiers in Microbiology \| www.frontiersin.org Frontiers in Microbiology \| www.frontiersin.org 9 Microbial Community of Indian Ocean Wang et al. DISCUSSION metC, cysteine-S-conjugate beta-lyase; metH, 5-methyltetrahydrofolate– homocysteine methyltransferase; serC, phosphoserine aminotransferase; serB, phosphoserine phosphatase; ltaE, threonine aldolase; glyA, glycine hydroxymethyltransferase; asdA, aspartate 4-decarboxylase; ALT, alanine transaminase; tyrB, aromatic-amino-acid transaminase; tyrC, cyclohexadieny/prephenate dehydrogenase; tyrB, aromatic-amino-acid transaminase; pheA2, prephenate dehydratase. +, present; –, absent. metC, cysteine-S-conjugate beta-lyase; metH, 5-methyltetrahydrofolate– homocysteine methyltransferase; serC, phosphoserine aminotransferase; serB, phosphoserine phosphatase; ltaE, threonine aldolase; glyA, glycine hydroxymethyltransferase; asdA, aspartate 4-decarboxylase; ALT, alanine transaminase; tyrB, aromatic-amino-acid transaminase; tyrC, cyclohexadieny/prephenate dehydrogenase; tyrB, aromatic-amino-acid transaminase; pheA2, prephenate dehydratase. +, present; –, absent. The picocyanobacterium Prochlorococcus was the most abundant photosynthetic phytoplankton in the oligotrophic oceans, and contributes to the stability, resilience and function of marine ecosystem with high species diversity and a wide range of environmental adaptability (Biller et al., 2015; Farrant et al., 2016; Kent et al., 2016). In this study, the major Prochlorococcus clades in the Indian Ocean belonged to HLII ecotypes, which is in accordance with the sample origins from surface water with high light intensity. A previous study also showed that most of the Indian Ocean Prochlorococcus sequences were distantly related to the HLII Prochlorococcus marinus spp. such as MIT9215 and MIT9301, which were also detected in our samples (Díez et al., 2016). Furthermore, HLII bins also dominate the Prochlorococcus communities of the surface waters of the Red Sea based on analysis of the rpoC1 sequences (Shibl et al., 2014). But the HLI, HLII, and HLVI clades of Prochlorococcus dominate in the surface water of the North Paciﬁc Ocean by clustering the ITS sequences (Larkin et al., 2016). Phylogenetic analysis of 504 core genes across global ocean samples indicate that the clades HLIII and HLIV signiﬁcantly dominate in the Equatorial Paciﬁc Ocean, and the clad HLI is abundant in the California Current and South Atlantic Ocean (Kent et al., 2016). Therefore, the ecotype structure of Prochlorococcus diﬀers among the oceanic regions. In this study, we constructed a phylogenetic tree based on 40 SCGs. This alternative method has been widely used in the clade classiﬁcation of bacterial genomes (Hug et al., 2016; Mende et al., 2017). The phylogenetic clades showed the same accuracy as the ITS sequence (Biller et al., 2014) when we examined them using 25 fully sequenced reference genomes with known ecotypes. We also examined the accuracy of COG0172 in distinguishing species diﬀerence among 25 known ecotype genomes before exploring the global distribution of Prochlorococcus. Frontiers in Microbiology \| www.frontiersin.org DISCUSSION TABLE 1 \| Distribution patterns of key enzymes of six amino acids synthesis pathways in Prochlorococcus metagenomes from the Indian Ocean. Amino acids Key enzymes Transporters Met metC metH – – + Ser serC serB – + – Gly ltaE – glyA + betT, betS: choline/glycine/proline betaine transport protein Ala asdA – ALT – alanine or glycine cation symporter Tyr tyrB tyrC – – + Phe pheA2 tyrB – + – metC, cysteine-S-conjugate beta-lyase; metH, 5-methyltetrahydrofolate– homocysteine methyltransferase; serC, phosphoserine aminotransferase; serB, phosphoserine phosphatase; ltaE, threonine aldolase; glyA, glycine hydroxymethyltransferase; asdA, aspartate 4-decarboxylase; ALT, alanine transaminase; tyrB, aromatic-amino-acid transaminase; tyrC, cyclohexadieny/prephenate dehydrogenase; tyrB, aromatic-amino-acid transaminase; pheA2, prephenate dehydratase. +, present; –, absent. TABLE 1 \| Distribution patterns of key enzymes of six amino acids synthesis pathways in Prochlorococcus metagenomes from the Indian Ocean. Prochlorococcus genomes. The Prochlorococcus sequences from the global ocean could be divided into two clades (Moore et al., 1998; Rocap et al., 2002). Our study indicated that the HL clade occupied most of the sequences and showed less divergence, but the LL clades showed more branches with various phylogenetic distance. Similar observations were found in Yan’s study (Yan et al., 2018). At this point, most samples are sourced mainly from surface water and less from the deep ocean. The highly diﬀerent phylogenetic distance of Prochlorococcus sequences from the deep ocean may reﬂect more diversity of Prochlorococcus genomes. These results illustrated that there was a considerable diversity of Prochlorococcus in the global ocean, but only a small portion can be cultured in the laboratory and a lot of unknown strains requires further excavation. The RDA analysis showed that the Prochlorococcus abundance was positively associated with temperature and pH. Previous studies have shown that the HLI and HLII clades of Prochlorococcus present diﬀerent optimal temperatures, indicating that temperature inﬂuences the distribution of Prochlorococcus (Zinser et al., 2007; Flombaum et al., 2013). The eﬀect of pH on Prochlorococcus growth has been less studied, but it directly impacts the availability of bicarbonate for photosynthetic reaction, which may aﬀect carbon ﬁxation and cell growth. The abundance of Prochlorococcus was also signiﬁcantly yet negatively correlated with nitrite concentration, suggesting the potential role of nitrite in Prochlorococcus growth. CONCLUSION The oligotrophic Indian Ocean is an understudied realm of the world’s oceans. This study comprehensively analyzed the microbial diversity and metabolic potential for N acquisition in the surface waters of the oligotrophic Indian Ocean using a metagenomic approach. Proteobacteria and Cyanobacteria dominated the microbial community but the functional composition of microbes exhibited a high level of gene diversity and functional redundancy across the Indian Ocean from the east to the west. Environmental factors such as temperature, phosphate, silicate, and pH played important roles in regulating microbial distribution in the Indian Ocean. Ammonium was an important nitrogen source for microbial community, while bacterial functional gene nifH was absent in the surface water of the Indian Ocean, indicating weak nitrogen ﬁxation in the Indian Ocean and other potential ammonium origins, which needs further study. The predominant cyanobacterial taxa Prochlorococcus presented high diversity but a simple ecotype. Moreover, the Prochlorococcus evolved diverse adaptive strategies to ambient N deﬁciency in the oligotrophic ocean. Interestingly, the gaps for speciﬁc amino acid biosynthesis pathways existed in Prochlorococcus, demonstrating that there could be some alternative ways to acquire some essential amino, and the potential roles of Prochlorococcus in the biogeochemical cycle of amino acids. Overall, this study facilitated our understanding DISCUSSION Our study elaborated nitrogen assimilation pathways in the Prochlorococcus population and Prochlorococcus genomes in the Indian Ocean, and identiﬁed the diﬀerences in utilization of inorganic and organic nitrogen sources among diﬀerent strains. This ﬁnding enables the potential to discover more unknown and uncultured strains from oceans. of microbes in the oligotrophic Indian Ocean, and serves as an important resource for gene capacity of microbes in future studies. AUTHOR CONTRIBUTIONS D-ZW, Y-YW, TJ, and XL established the concept of the study. YBG, Z-XX, and L-FK collected and processed the samples. Y-YW, S-LL, G-LL, and S-HG performed the bioinformatics analyses. Y-YW, S-LL, and P-FZ performed the data analysis. Y-YW wrote the draft. D-ZW, HL, LG, M-LS, S-LL, S-KS, and G-YF revised and edited the manuscript. All the authors have discussed the results, read and approved the contents of the manuscript. DATA AVAILABILITY STATEMENT The 17 fastq ﬁles of SE reads were deposited in the CNSA (https://db.cngb.org/cnsa/) of CNGBdb with accession code CNP0000411. The 3 fastq ﬁles of PE reads were submitted to the NCBI SRA database with accession number PRJNA450884 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA450884). It has been known for decades that most of the free-living bacteria can synthesize 20 kinds of amino acids, but some bacteria still have gaps in their biosynthesis pathways of amino acids (Price et al., 2018). Our study showed that Prochlorococcus could not make all amino acids by themselves based on their genetic content and might need to uptake some of them from the ambient environment instead. The gaps in de novo synthesis pathways of six diﬀerent amino acids implied a special model for amino acid utilization in the Prochlorococcus population. Moreover, the distribution pattern of key enzymes involved in N and amino acid metabolism pathways in the metagenome of the Prochlorococcus population was validated by the Prochlorococcus genomes identiﬁed, demonstrating the reliability of our results. In general, our ﬁndings illustrated the potential capacity of Prochlorococcus to assimilate nitrogen in the Indian Ocean, which implied the adaptation of Prochlorococcus to ambient N deﬁciency in the oligotrophic ocean. DISCUSSION It was demonstrated to be suﬃcient to discern the phylogenetic relation among The microbial functional genes involved in nitrogen metabolism indicated that bacteria in the oligotrophic ocean could assimilate N through diﬀerent routes and they preferred urea, ammonia and nitrile, which need low energy to produce ammonia. The bacterial functional gene nifH, which is responsible for nitrogen ﬁxation was absent in the surface water of the Indian Ocean and a similar phenomenon was also observed in the west Paciﬁc Ocean (Li Y.-Y. et al., 2018), indicating that bacteria in the Indian Ocean surface waters lacked the ability to ﬁx nitrogen. Ammonia assimilation of Prochlorococcus was explored in this study. The Prochlorococcus population can assimilate urea, ammonia, nitrite, nitroalkane, cyanate, and nitrile as nitrogen sources for cell growth, indicating that Prochlorococcus had evolved diverse adaptive strategies to ambient N deﬁciency in the oligotrophic Indian Ocean. The abundances of key enzymes involved in DON assimilation were higher than those involved in DIN assimilation in the Prochlorococcus population, implying that Prochlorococcus might prefer the organic N source in the oligotrophic Indian Ocean. The transcripts of key enzymes involved in the utilization of cyanate, urea and ammonia are also detected in the Prochlorococcus genomes under N stress condition (García-Fernández et al., 2004; Kamennaya and Post, 2011). To date, no Prochlorococcus isolate is able to utilize molecular N due to the lack of nifH genes, which concurs with our ﬁndings. The ability of nitrate assimilation is found only in a small portion of the Prochlorococcus genomes, such as SB and MIT0604, which is consistent with the ﬁndings of a recent study (Berube et al., 2019). They can grow on nitrate as the sole nitrogen source (Martiny et al., 2009; Berube et al., 2015). In our study, the gene encoding NarB catalyzing nitrate to nitrite was not detected in February 2021 \| Volume 12 \| Article 518865 10 Microbial Community of Indian Ocean Wang et al. the Prochlorococcus population although it was found in 14 of the identiﬁed 54 Prochlorococcus genomes, indicating the limitation in assembling full sequences from metagenome data and the importance of isolation and identiﬁcation of single bacterial strain from the natural environment, which would help us to unravel more novel and divergent metabolic pathways. FUNDING This work was supported by funding from the National Natural Science Foundation of China (41425021), the International S&T Cooperation (2016YFE0122000), Shenzhen Municipal Government of China (No. JCYJ2015015162041454), the Guangdong Provincial Key Laboratory of Genome Read and Write (2017B030301011), and the Danish National Research Foundation (DNRF137). This work was supported by funding from the National Natural Science Foundation of China (41425021), the International S&T Cooperation (2016YFE0122000), Shenzhen Municipal Government of China (No. JCYJ2015015162041454), the Guangdong Provincial Key Laboratory of Genome Read and Write (2017B030301011), and the Danish National Research Foundation (DNRF137). Frontiers in Microbiology \| www.frontiersin.org REFERENCES Prochlorococcus: the structure and function of collective diversity. Nat. Rev. Microbiol. 13:13. doi: 10.1038/nrmicro3378 Langmead, B., and Salzberg, S. L. (2012). Fast gapped-read alignment with Bowtie 2. Nat. Methods 9:357. doi: 10.1038/nmeth.1923 Larkin, A. A., Blinebry, S. K., Howes, C., Lin, Y., Loftus, S. E., Schmaus, C. A., et al. (2016). Niche partitioning and biogeography of high light adapted Prochlorococcus across taxonomicranks in the North Paciﬁc. ISME J. 10, 1555– 1567. doi: 10.1038/ismej.2015.244 Buchﬁnk, B., Xie, C., and Huson, D. H. (2015). 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Supplementary Figure 3 \| Microbial taxonomic composition and predominant bacterial phylum in the Indian Ocean surface water. (A) Taxonomic composition. (B) The predominant bacterial phylum. Others represented bacterial abundance lower than 0.1%. Supplementary Figure 4 \| The heat map of physicochemical properties from oceanic samples. Supplementary Figure 4 \| The heat map of physicochemical properties from oceanic samples. Supplementary Figure 5 \| The distribution pattern of the detected Prochlorococcus genomes among sampling stations of the Indian Ocean. Frontiers in Microbiology \| www.frontiersin.org Frontiers in Microbiology \| www.frontiersin.org February 2021 \| Volume 12 \| Article 518865 11 Microbial Community of Indian Ocean Wang et al. REFERENCES Metagenomic exploration of viruses throughout the Indian Ocean. PLoS One 7:e42047. doi: 10.1371/journal.pone.0042047 Qin, J., Li, R., Raes, J., Arumugam, M., Burgdorf, K. S., Manichanh, C., et al. (2010). 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Oceanogr. 31, 998–1009. doi: 10.4319/lo.1986.31.5.0998 Qian, G., Wang, J., Kan, J., Zhang, X., Xia, Z., Zhang, X., et al. (2018). Diversity and distribution of anammox bacteria in water column and sediments of the eastern Indian Ocean. Int. Biodeterior. Biodegr. 133, 52–62. doi: 10.1016/j.ibiod.2018. 05.015 Williamson, S. J., Allen, L. Z., Lorenzi, H. A., Fadrosh, D. W., Brami, D., Thiagarajan, M., et al. (2012). Frontiers in Microbiology \| www.frontiersin.org REFERENCES Structure and function of the global ocean microbiome. Science 348:1261359. Copyright © 2021 Wang, Liao, Gai, Liu, Jin, Liu, Gram, Strube, Fan, Sahu, Liu, Gan, Xie, Kong, Zhang, Liu and Wang. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Copyright © 2021 Wang, Liao, Gai, Liu, Jin, Liu, Gram, Strube, Fan, Sahu, Liu, Gan, Xie, Kong, Zhang, Liu and Wang. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Sunagawa, S., Mende, D. R., Zeller, G., Izquierdo-Carrasco, F., Berger, S. A., Kultima, J. R., et al. (2013). Metagenomic species proﬁling using universal phylogenetic marker genes. Nat. Methods 10, 1196–1199. doi: 10.1038/nmeth. 2693 Thompson, L. R., Williams, G. J., Haroon, M. F., Shibl, A., Larsen, P., Shorenstein, J., et al. (2017). Metagenomic covariation along densely sampled environmental gradients in the Red Sea. ISME J. 11:138. doi: 10.1038/ismej.2016.99 February 2021 \| Volume 12 \| Article 518865 Frontiers in Microbiology \| www.frontiersin.org 13
https://openalex.org/W2010652451	https://zenodo.org/records/2310845/files/article.pdf	English	null	THE SIN OF TREATING SYMPTOMS	JAMA	1,918	public-domain	2,482	THE SIN OF TREATING SYMPTOMS H. S. WARD, M.D. BIRMINGHAM, ALA. At frequent intervals since the age of puberty, she had attacks of nausea and vomiting called indigestion, headaches, neurosis, etc. Finally her ureters were catheterized and she was found to have calculi in both kidneys. The stones were removed, and the operation gave much relief. The omission may be due to the lack of using one's own faculties. This fault has been hammered so long that one rarely sees the glaring errors once so com¬ mon. The omission is more often the refusal to accept, or the inability to secure, outside help. I once heard of a physician who refused consultation, saying that a patient who had him needed no other physician. Such an egotist is a menace to the community. The men who refuse consultation, fear for their own repu¬ tations and care very little for the welfare of their patients. Group 2.—The Wassermann test alone will not aid in diagnosing all our indefinite and latent cases of syphilis. Case 3.—X., a man, aged 41, who had lived a strenuous and dissipated life without any previous history of syphilis, sud¬ denly began to have jacksonian and epileptic convulsions. The blood Wassermann reaction was negative. He was treated for epilepsy. But the irregular pupils, the slightly slurring speech, the tremor, just discernible, about the lips, and a pleocytosis in the spinal fluid definitely labeled him a victim of early general paresis. patients. Jealousy has wrecked many a small mind. The real physician, the one who has only the good of his patient in mind, never fears roentigen-ray findings, laboratory results, dental examination, or specialists' reports. The specialist is with us to stay, and is most useful, but he must not be permitted to subsidize the whole patient to aggrandize the disease of one partic¬ ular organ. It has long been recognized that epileptic seizures appearing in the adult are often precursors of an early general paresis. Even focal infection and a neurosis due to menopausic changes will not always be sufficient. o ga The clinician must not allow the laboratory or the specialist to make his diagnoses ; if so, his days of usefulness are gone. These factors are only aids. One cannot always delay until the pathognomonic symptom appears. THE SIN OF TREATING SYMPTOMS H. S. WARD, M.D. BIRMINGHAM, ALA. THE SIN OF TREATING SYMPTOMS H. S. WARD, M.D. BIRMINGHAM, ALA. g p Case 1.—Mrs. W., aged 35, had a diseased heart, had been an invalid for many years, and was hysterical and unmanage¬ able. She had attacks of vomiting and pain in the upper left side of the abdomen. These occurred every few weeks and she had been treated by some of the best physicians in Bir¬ mingham without obtaining relief. She was considered a neurotic. She had gone to one of our leading medical cen¬ ters, where she had been given a rest cure, with some tem¬ porary relief. After changing physicians many times, she fell into my hands. A series of roentgenograms revealed ptosed viscera. One of our best surgeons removed several feet of her colon, an operation followed by temporary relief, only to see her relapse into her old attacks again. She was then seen by many surgeons and internists, all of whom assured her that she was a neurotic and that she should "quit her foolishness." Finally she found a surgeon who had her ureters catheterized, discovered a suppurating kidney, and removed it. This took place several years ago. She is now well. The seven deadly sins were enumerated before the days of modern medicine or specialism. The sin of omission would then have been classed as venial; but now, omission is often deadly to our patients, and might be classed by modern moralists as unpardonable.f might by unpardonable. This paper is intended to report some groups of cases that have been improperly diagnosed by observ- ing leading symptoms without taking the patient as a diagnostic problem and viewing him as a whole. Med- icine is no longer a one man's job\p=m-\itis team work. It is, indeed, rare for one to be able to diagnose an obscure case alone. Team work with its proper limita- tions is our hope for the best results. Diagnostic units are now essential. There is much truth in the saying that only the very poor and the very rich get proper medical attention. The poor may go to a well organ- ized hospital, where a department chief collects all required data, proper diagnosis is made, and required treatment instituted. Case 2.—Mrs. D., aged 32, married, with two children, had definite deformities following a multiple neuritis, com¬ plicated by a severe attack of typhoid. THE SIN OF TREATING SYMPTOMS H. S. WARD, M.D. BIRMINGHAM, ALA. One who waits for the laboratory to find tubercle bacilli in the sputum before diagnosing pulmonary tuberculosis, will realize that he has waited too long to be of real value to his patient. The patient must be viewed as a whole, the laboratory and specialists' findings being subsidiary to one grand summing up. Who shall weigh these findings in the balance and be the final arbiter ? The one who should be most capable is the practitioner or internist. Case 4.—Mrs. S., aged 43, had found her health beginning to fail. Focal infection was suggested. The tonsils were removed and the teeth were gone over without improvement. The ailment was then diagnosed as a neurosis due to meno¬ pausic changes. When I first saw her, there were double choked disks and aphasia. The spinal fluid and the blood showed a negative Wassermann reaction. A diagnosis of temporosphenoidal tumor was made. At operation she was found to have multiple gliomas. Group j.—Prolonged rest and polypharmacy are no longer sufficient, if one is to secure the best results in definite chorea. Case 5.—H, a schoolgirl, aged 17, had her tonsils removed several years ago. She had been rather a delicate girl. She came to me with definite chorea. The chorea without tonsils was indeed puzzling. Her teeth were irregular and she had been wearing a brace to correct dental deformity. On exam¬ ination it was found that she had definite pyorrhea. Rest in bed and the curing of the pyorrhea have restored her to health. capab e p act t o e If in these case reports, some physician should recognize an old friend, I beg of him not to take it as a personal criticism. The intent is to point out one of those broad truths applicable to us all, and is in no sense personal or intended as petty criticism. diagnose a diseased kidney and thus cure a long suffer¬ ing and nervous patient. diagnose a diseased kidney and thus cure a long suffer¬ ing and nervous patient. CHRONIC SEPTICEMIC ENDOCARDITIS WITH SPLENOMEGALY TREATMENT BY SPLENECTOMY DAVID RIESMAN, M.D. PHILADELPHIA Case 12.—Mrs. C, housewife, aged 62, who had been a rheumatic for years, and was recently found to have high blaod pressure, was suddenly seized with anginoid attacks. After about three weeks of waiting for death to claim her and of exhausting a large supply of amyl nitrite pearls, she was sent to a hospital and found to have abscessed teeth and diseased tonsils. These foci were removed, and she became a useful woman. Her intercostal neuralgia is now a harrowing memory to a very good doctor. In the face of a disease like cancer or sarcoma we accept our helplessness with resignation; but we chafe against our helplessness in so-called malignant endo- carditis, for we feel that we should be able to master it, the disease being an infective process due to well known bacteria, which in other circumstances may be successfully combated. Dr. Libman has shown that patients suffering from this type of endocarditis may become bacteria free and yet succumb to the disease. If death were an early event, the failure of our efforts might be more readily understood; but the affection may last for months or even for one or two years, so that ample time exists for treating it, and yet nearly every case terminates fatally. I g g Case 13.—D., aged 45, a robust man of good habits, had severe rheumatic pains and intense, diffuse bronchitis, with asthmatic breathing. He was unable to lie down at night. On examination, he was found to have pyorrhea, with many abscessed teeth. Extraction of the teeth and treat¬ ment of the gums for a short time, followed by two weeks at the seashore, completely restored him. , p y Case 14.—L., aged 17, a clerk, had been troubled with a number of painful abscesses. An abscessed tooth was ex¬ tracted. His general health improved and no furunculosis has since occurred. . e e y fatally. I shall not go into detail regarding the symptoms of the affection, with which the writings of Osler, Dock and Libman have made us familiar. The outstanding features are fever of long duration, which may resem- ble that of tuberculosis or malaria ; anemia of varying intensity, sometimes so severe as to suggest one of the primary anemias; pains in the joints; petechial spots on the skin and mucous membranes ; and a heart mur¬ mur. MISTAKES IN DIAGNOSIS His tonsils were removed and he was soon able to go on with his work without any further thyroid disturbance. g Then comes the practical problem, What are we going to do about it? Prevention, by teaching oral hygiene, will help the future generation. But our own patients must be taken care of. Shall we deal with this pathologic condition radically or conservatively? Many deaths have been reported from too radical treat¬ ment. Apparently the best results are obtained by putting the patient to bed, clearing up the pathologic condition a little at each sitting, allowing from three to ten days between treatments, according to the amount of reaction and recuperation of the patient. Group 5.—Let us beware of that old hobbyhorse we have so often ridden—a nervous breakdown from overwork. If we look closely, we shall find something else to account for the patient's condition. Case 10.—Dr. D., aged 39, after a rather careful examina¬ tion, had been sent to the seashore with his wife to recover from "overwork." His nervousness had increased and when I saw him, general paresis was easily diagnosed. He after¬ ward died in the insane asylum. ecupe at o pat e t Is oral infection a passing fad? Perhaps we are reading too much into it. It is certainly easy to become too enthusiastic. To me it is one of the pres¬ ent problems in medicine, extending into all the spe¬ cialties. We should, however, remember the plea to view the whole patient as a diagnostic problem. If we work and watch while waiting, oral infection will find its proper place in medicine. y Case 11.—B., a traveling man, aged 41, was considered overworked, notwithstanding his robust physical condition. The symptoms were irregular pupils, insomnia, depressed spirits and a ++ H—h Wassermann reaction. It was decided best to send him to an institution for treatment and con¬ finement. Group 6.—The last and largest group includes cases of oral infection, pyorrhea, and abscessed and carious teeth. Everybody is talking about oral infection and, perhaps, exaggerating its importance. MISTAKES IN DIAGNOSIS Case 6.—L., aged 9, a schoolboy, came into my office on crutches. The right side suggested a mild hemiplegia. The case was easily diagnosed as chorea, with chronically dis¬ eased tonsils. Tonsillectomy and rest in bed cured the hemiplegia and chorea. Group i.—Many inoffensive appendixes have been operated on, many normal tubes and ovaries have been sacrificed and many useful gallbladders drained ; many ptosed viscera have been stitched up or colons removed, and many healthy stomachs have had a new opening, only for some painstaking surgeon later to p g Case 7.—S., a schoolgirl, aged 16, had often suffered during the winter from catarrh and colds. She was found to be aded From: http://jama.jamanetwork.com/ by a University of Arizona Health Sciences Library User on 06/02/201 suffering from classical chorea. Removal of the tonsils and rest in bed soon restored her to perfect health. suffering from classical chorea. Removal of the tonsils and rest in bed soon restored her to perfect health. Case 17.—C, a man, aged 67, had been troubled with a number of furuncles. He was found to have almost the same symptoms as the patients in the two foregoing cases. All his teeth had to be removed. He insisted on trying to attend to some business, which proceeding made his con¬ valescence slow. Group 4.—Other things may suffice to relieve hyper- thyroidism, rather than resort to the surgery of the thyroid gland. In the Hillman Hospital there are many of the adult and aged "down and out" class, who, in times past, have been healthy and useful citizens. As a rule their mouths and teeth have been grossly neglected. Their teeth are irregular, loose, carious and broken, and set in gums seething with pus. I have often asked myself the question, "Is there any connection between oral infection and being 'down and out'?" Th Case 8.—Mrs. B., a housewife, aged 23, mother of two children, had had goiter for several years. \Vhen I saw her, the symptoms were tachycardia, tremors, an enlarged thyroid, von Graefe's sign, sweating, emotionalism, insomnia, a phobia of being alone, etc. The tonsils, which had given trouble for years, were removed. After two weeks in bed and a trip to the country for six weeks, she was markedly improved. p Case 9.—L., a barber, aged 20, had the ordinary symptoms of hyperthyroidism. He had suffered with tonsillitis every winter since childhood. CHRONIC SEPTICEMIC ENDOCARDITIS WITH SPLENOMEGALY TREATMENT BY SPLENECTOMY DAVID RIESMAN, M.D. PHILADELPHIA Albuminuria is common, and in later stages the signs of a true nephritis may appear. Abdominal pains, sometimes sudden in onset and suggesting embolie processes in the spleen or kidneys, are not infrequent. Cough and insomnia are often very troublesome. From the diagnostic point of view, the Case 15.—O., a man, aged 66, who was in the insurance business, had a well defined arrhythmia. The heart sounds were almost inaudible, and the blood pressure was low. He experienced dyspnea on the slightest exertion, and his liver was enlarged. His spirits were markedly depressed. He was found to have five abscessed teeth, which were removed at two sittings. He has greatly improved, and the normal heart condition has been completely restored. p y Case 16.—C, a man, aged 50, suffered from asthenia, from an intense depression of spirits, that was almost melancholia, from marked insomnia, and loss of appetite. He was prac¬ tically bedridden. A number of severely abscessed teeth were removed, and he is making a slow but definite recovery. aded From: http://jama.jamanetwork.com/ by a University of Arizona Health Sciences Library User on 06/02/201
https://openalex.org/W4226207147	https://zenodo.org/record/6458417/files/IJET-V8I2P5.pdf	English	null	STATISTICAL ANALYSIS OF FREQUENCY DOMAIN FILTERS FOR IMAGE DENOISING	Zenodo (CERN European Organization for Nuclear Research)	2,022	cc-by	3,182	2. Proposed Method Technology occupies an essential role in the 21st century. The data revolution has vastly increased the scope of digital devices in our lives. The use of digital images has massively increased due to its cost-effectiveness and easy portability. However, digital images have some disadvantages. It can get corrupted by noise, which is an unwanted signal during any stage, be it image acquisition, segmentation or representation. Research in the field of digital image processing has determined three significant forms of noise that can taint an image. The three major types of noise are Gaussian noise, speckle noise and salt and pepper noise. In this paper, we propose to compare various image denoising techniques that remove noise from an image. The images that have been denoised using each method can be compared based on some parameters and find the best image denoising technique with the best performance parameters. The following diagram indicates the work that we propose to do. Fig. 1. Illustration diagram of the proposed system The percentage of corrupted pixels quantifies noise. Corrupted pixels are moreover set to the maximum or have single bits. There are several algorithms to remove salt and pepper noise [1,2,3]. The adaptive filter [4] gave noticeable results in the statistical analysis of various denoising techniques in MR images. Image retrieval [5,6] turned out to be an appealing field of interest. The convolution techniques instigated in [7] use kernels for filtering. Liu [8] explored the denoising algorithm employing a Wiener filter. Burger [9] proclaimed the impact of total variation algorithm in the reconstruction of images. Salloum [10] endorsed adaptive wavelet for data compression. Fig. 1. Illustration diagram of the proposed system Wang database has been used as it has a collection of 1000 random images from 10 different categories of images. Any image can be randomly selected from the database as an input for processing. The type of noise which must be tested can also be randomly chosen from Gaussian, speckle or salt and pepper noise. The input image is intentionally corrupted with the chosen noise to obtain a noisy image and test all the image denoising techniques for their performance and capability. This paper commends various techniques for effective noise removal from any image. The methods incorporate Mean filter [7], Median and Wiener filter [8], Bilateral filter, Fourier transform, Haar wavelet, Daubechies wavelet and Curvelet transform. STATISTICAL ANALYSIS OF FREQUENCY DOMAIN FILTERS FOR IMAGE DENOISING 1D. Madhavi, 2N. Jyothi 1Associate Professor, 2Associate Professor 1Department of EECE, 1GITAM Deemed to be University, Visakhapatnam, India Abstract – With the advent of the digital world, the use of digital images has become widespread. During the process of image acquisition, image contamination by noise becomes an inevitable part of the image, leading to a significant reduction in quality. Traditionally, filters remove noise from images. Technological advances in the arena of image processing have led to growth of more efficient techniques of image denoising. These techniques make use of wavelets and Curvelet transforms, which can be blended with known parts of noisy image to estimate the unknown parts of the image better. In this paper, we apply various image denoising techniques to a random image that is corrupted by either Gaussian noise, speckle noise or salt and pepper noise. The evaluation results in terms of Mean Squared Error, Peak Signal to Noise Ratio, Structural Similarity Index and Computation time will help to decide the best technique that is used for denoising an image distorted by noise. Keywords: Image denoising techniques, Curvelet, discrete wavelet transform, filters , national Journal of Engineering and Techniques - Volume 8 Issue 2, March 202 national Journal of Engineering and Techniques - Volume 8 Issue 2, March 202 Keywords: Image denoising techniques, Curvelet, discrete wavelet transform, filters , 2.2.2 Median filter It is a sliding-window filter in spatial frequency domain which replaces median value with mean of all pixel values in window function. 2.2.4 Bilateral filter Salt and pepper noise It smooths images while maintaining edges, using non- linear blend of adjacent pixel values. It performs both range and domain filtering and replaces a bright pixel with an average of nearby bright pixels(while ignoring dark pixels). It returns a dark pixel with an average of nearby dark pixels(while ignoring bright pixels). It is also termed impulse noise or spike noise. An image will have dark pixels in vivid regions and vivid pixels in dark areas if it is corrupted with salt and pepper noise. Speckle noise It is inherently present in the image. Random values augmented by pixel values can model this noise. Consequently, it is labeled multiplicative noise. Speckle noise is significant obstacle in radar applications. International Journal of Engineering and Techniques - Volume 8 Issue 2, March 2022 The original input image is denoised using each of the image denoising techniques, and the quality of images can be compared using the parameters of the denoised image as peak signal-to-noise ratio, mean squared error and structural similarity index. The time taken to compute each denoised image is also available for comparison. It is a sliding-window filter in spatial frequency domain which replaces center value with mean of all pixel values in window function. 2.2.3 Wiener filter Gaussian noise It figures a statistical assessment of an anonymous signal using an associated signal as input besides filtering that known signal to generate the approximate as an output. It arises during the process of acquisition of digital images. It has a probability density function identical to normal Gaussian distribution, i.e., the values that can be taken by the noise are Gaussian-distributed. 2.1 Types of Noise Noise is unwanted data in an image. There are various ways for the introduction of noise. Some of them include the acquisition of the digital image, the film grain when scanning an image from a photograph. 2.2 Filters • Obtain clean coefficients from noisy ones through estimation using hard thresholding or soft thresholding. • Obtain clean coefficients from noisy ones through estimation using hard thresholding or soft thresholding. Image denoising is essential to ensure a minimum acceptable quality of an image. Denoising of images is distinct from enhancement. Image enhancement is an unbiased process, while image denoising is a skewed process. Many denoising algorithms are applied to generate the best estimate of an image using a noisy image. •Inverse Wavelet Transform is used to attain denoised image from the clean coefficients. 2.2.5 Fourier transform filters Fourier transform filters generally specify the frequency and not time, and hence they are not preferred. It can give the frequency components existing in a signal but cannot capture the time at which the frequency components occur. Fig. 2. Illustration of original image and image with types of noise 2 2 Filt 2. Proposed Method The quality parameters of the denoised images obtained by various techniques are analyzed and arrive at the best denoising technique. Image denoising makes use of spatial domain filtering or transform domain filtering. Filtering in spatial domain involves use of linear filters or non-linear filters that include mean filter, median filter, Wiener filter along with Bilateral filter. Filtering in transform domain have Fourier transform, Haar transform, Daubechies wavelet transform, and Curvelet transform. ISSN: 2395-1303 Page 20 http://www.ijetjournal.org 2.2.5 Curvelet transform filters Curvelets are pertinent basis for characterizing images which are smooth except for singularities beside smooth curves, where objects in the image have a least length scale and curves 2.2.5 Wavelet transform filters A tiny wave-like fluctuation with an amplitude that commences at zero, boosts and declines back to zero is termed wavelet. It has its energy focused on time along with frequency. Wavelets have identifiable properties that make them incredibly beneficial for image processing. Wavelets can be merged with well-known sections of a noisy signal to extricate the information from unidentified servings through convolution. All wavelet transforms generally involve the following three steps Fig. 2. Illustration of original image and image with types of noise •Forward Wavelet Transform is employed to obtain wavelet coefficients. •Forward Wavelet Transform is employed to obtain wavelet coefficients. 2.2 Filters 3. Evaluation Results The noisy images are being denoised using a variety of image denoising techniques. Therefore, some parameters are required to judge the quality and acceptability of the denoised image. The parameters for evaluation are • Mean Squared Error (MSE) • Peak Signal-to-Noise Ratio (PSNR) • Structural Similarity Index (SSI) For image corrupted with Gaussian noise, among all the spatial filters that we have used Bilateral filter is best and efficient because it has a least MSE value of 340.45, highest PSNR value of 22.81 and highest SSI value of 0.48007. • Computation Time (CT) 2.2.1 Mean filter ISSN: 2395-1303 Page 21 http://www.ijetjournal.org International Journal of Engineering and Techniques - Volume 8 Issue 2, March 2022 International Journal of Engineering and Techniques - Volume 8 Issue 2, March 2022 have bounded curvature. This holds for caricatures, geometrical drawings, and wording. The edges seem gradually straight as one whizzes in on such images. Curvelets take benefit by describing the better resolution curvelets to be more stretched than the lower resolution curvelets. Curvelets are preferred to wavelets because command 'tic' begins the timer while command 'toc' closes the timer, and display the lapsed time (in seconds). Table 1. Comparison of an optimal solution for Gaussian Noisy Image Table 1. Comparison of an optimal solution for Gaussian Noisy Image p p y g MSE PSNR SSI CT Noisy Image 624.92 20.173 0.36929 0 Linear Filter 6904.4 9.7396 0.10736 0.22866 Median Filter 1216.6 17.279 0.45462 0.26344 Wiener Filter 840.85 18.884 0.4665 0.41511 Bilateral Filter 340.45 22.810 0.48007 0.24118 Fourier Transform 624.78 20.174 0.36933 0.15731 Haar Transform 240.6 24.318 0.65024 0.23836 Daubechies wavelet Transform 246.57 24.211 0.68629 0.35277 Curvelet Transform 128.18 27.052 0.78773 0.26786 •It is an optimally sparse portrayal of objects along with edges. •It provides for optimal image reconstruction. Peak Signal-to-Noise Ratio It is ratio stuck between extreme possible value (power) of signal and power of misleading noise that alters quality of its interpretation. It is articulated in decibels and calculated using the formula: Fig. 3. Computational results with spatial domain filters for Gaussian Noisy image When compared with all other transform filters, curvelet When compared with all other transform filters, curvelet transform is best because it has the least MSE value of 128.18, highest PSNR value of 27.052 and highest SSI value of 0.78773. 𝑃𝑆𝑁(𝑖𝑛 𝑑𝐵) = 10 log10 ( 𝑀𝐴𝑋2 ) (2) 𝑀𝑆𝐸 where MAX stands for extreme possible pixel value of image. For 8-bit images, MAX = 28-1 = 255, and MSE is mean-squared error. 𝑃𝑆𝑁(𝑖𝑛 𝑑𝐵) = 10 log10 ( 𝑀𝐴𝑋2 ) (2) 𝑀𝑆𝐸 (2) transform is best because it has the least MSE value of 128.18, highest PSNR value of 27.052 and highest SSI value of 0.78773. where MAX stands for extreme possible pixel value of image. For 8-bit images, MAX = 28-1 = 255, and MSE is mean-squared error. Fig. 4. Computational results with transform domain filters for Structural Similarity Index Structural Similarity Index It measures the resemblance between two images. By taking the original image as a reference, the SSIM indicates the degree of similarity stuck between noisy/denoised image and the original image. The value of SSIM is always between 0 and +1. The value 1 is possible only with identical sets of data, (i.e. same images). A value of 0 signifies no structural resemblance. More the value of SSIM, clearer is the quality of the denoised image. Mean Squared Error Fig. 3. Computational results with spatial domain filters for Gaussian Noisy image When compared with all other transform filters, curvelet transform is best because it has the least MSE value of 128.18, highest PSNR value of 27.052 and highest SSI value of 0.78773. Fig. 3. Computational results with spatial domain filters for Gaussian Noisy image It represents the middling of squares of errors amid original image and noisy/denoised image. The error is quantity with which the values of original image vary from noisy/denoised image. Given original m x n image I combined with its approximation K, mean squared error can be estimated using: 𝑀𝑆𝐸 = 1 ∑𝑚−1 ∑𝑛−1[𝐼(𝑖, 𝑗) − 𝐾(𝑖, 𝑗)]2 (1) 𝑚𝑛 𝑖=0 𝑗=0 (1) Computation Time The computation time of each technique is calculated in MATLAB using the timer that is present in MATLAB. The Fig. 4. Computational results with transform domain filters for Fig. 4. Computational results with transform domain filters for ISSN: 2395-1303 Page 22 http://www.ijetjournal.org International Journal of Engineering and Techniques - Volume 8 Issue 2, March 2022 International Journal of Engineering and Techniques - Volume 8 Issue 2, March 2022 Gaussian Noisy image Gaussian Noisy image Fig. 5. Graph of evaluation parameters for removal of Gaussian noise using various filters Fig. 7. Computational results with transform domain filters for Salt and pepper noisy image Fig. 8. Graph of evaluation parameters for removal of Salt and pepper noise using various filters Fig. 7. Computational results with transform domain filters for Salt and pepper noisy image Fig. 5. Graph of evaluation parameters for removal of Gaussian noise using various filters Fig. 7. Computational results with transform domain filters for Salt and pepper noisy image Table 2. Comparison of an optimal solution for Salt and Pepper Noisy Image Table 2. Comparison of an optimal solution for Salt and Pepper Noisy Image Table 2. Comparison of an optimal solution for Salt and Pepper Noisy Image MSE PSNR SSI CT Noisy Image 1247.3 17.171 0.5311 0 Linear Filter 2824.1 13.622 0.40994 1.1773 Median Filter 2719.3 13.786 0.32027 1.3707 Wiener Filter 1804.7 15.567 0.31499 2.0568 Bilateral Filter 1252.2 17.154 0.50507 1.1745 Fourier Transform 1243.9 17.183 0.53131 0.40214 Haar Transform 1196.6 17.351 0.49437 0.68565 Daubechies wavelet Transform 670.25 19.868 0.59369 0.84875 Curvelet Transform 1001.3 18.125 0.53493 0.79413 Fig. 8. Graph of evaluation parameters for removal of Salt and pepper noise using various filters Fig. 8. Graph of evaluation parameters for removal of Salt and pepper noise using various filters For image distorted with salt and pepper noise, among all the spatial filters that we have used Bilateral filter is best and efficient because it has a least MSE value of 1252.2, highest PSNR value of 17.154 and highest SSIM value of 0.50507. For image distorted with salt and pepper noise, among all the spatial filters that we have used Bilateral filter is best and efficient because it has a least MSE value of 1252.2, highest PSNR value of 17.154 and highest SSIM value of 0.50507. Fig. 8. Graph of evaluation parameters for removal of Salt and pepper noise using various filters Fig. 6. Acknowledgements The author thanks the evaluators for their valued assessment and recommendation of this manuscript. When compared with all other transform filters, curvelet transform is best because it has the least MSE value of 285.2, highest PSNR value of 23.579 and highest SSI value of 0.63202. 4. Conclusion Fig. 9. Computational results with spatial domain filters for Speckle noisy image A random image from Wang database is distorted through either Gaussian noise or Salt and Pepper noise or Speckle noise. Then spatial and transform domain filters have been used for denoising the image. The quality parameters for evaluation unveiled the most optimum denoising technique. It is evident that in the case of Gaussian noise and speckle noise, Curvelet transform outperforms all other image denoising techniques, and Salt and pepper noise is most effectively removed by Debauchies wavelet transform. In the future, Deep Learning may generate a deep neural network that has better image denoising capability concerning any noise. Fig. 9. Computational results with spatial domain filters for Speckle noisy image International Journal of Engineering and Techniques - Volume 8 Issue 2, March 2022 of 19.858 and highest SSI value of 0.53618. Computation Time Computational results with spatial domain filters for Salt and pepper noisy image Table 2. Comparison of an optimal solution for Speckle Noisy Image Table 2. Comparison of an optimal solution for Speckle Noisy Image MSE PSNR SSI CT Noisy Image 912.36 18.529 0.47336 0 Linear Filter 8516.6 8.8281 0.16013 0.54458 Median Filter 1414.2 16.626 0.23841 0.46556 Wiener Filter 933.1 18.432 0.3176 0.76087 Bilateral Filter 671.79 19.858 0.53618 0.48738 Fourier Transform 911.9 18.531 0.47333 0.35465 Haar Transform 617.11 20.227 0.45127 0.44084 Daubechies wavelet Transform 344.98 22.753 0.54309 0.56682 Curvelet Transform 285.2 23.579 0.63202 0.92741 Fig. 6. Computational results with spatial domain filters for Salt and pepper noisy image When compared with all other transform filters, the Daubechies wavelet transform is best because it has the least MSE value of 670.25, highest PSNR value of 19.868 and highest SSI value of 0.59369. For image corrupted with Speckle noise, among all the spatial filters that we have used Bilateral filter is best and efficient because it has a least MSE value of 671.79, highest PSNR value ISSN: 2395-1303 Page 23 http://www.ijetjournal.org International Journal of Engineering and Techniques - Volume 8 Issue 2, March 2022 References Fig. 10. Computational results with spatial domain filters for Speckle noisy image Fig. 11. Graph of evaluation parameters for removal of Speckle noise using various filters Fig. 10. Computational results with spatial domain filters for Speckle noisy image [1] Thanh, D.N.H., Hai, N.H., Prasath, V.B.S. et al. A two-stage filter for high-density salt and pepper denoising. Multimed Tools Appl 79, 21013–21035 (2020). [2] Alaoui, N., Mitiche A., Houda A.B., and Mitiche, L. Effective hybrid genetic algorithm for removing salt and pepper noise. IET Image Processing, 14(2), 289-296, (2020). [3] Halder A., Halder S., Chakraborty S., and Sarkar A. Statistical Salt-and-Pepper Noise Removal Algorithm. International Journal of Image and Graphics, 19(01), 1950006 (2019) [4] Setu Garg, Ritu Vijay, Shabana Urooj, " Statistical Approach to Compare Image Denoising Techniques in Medical MR Images," Procedia Computer Science, vol.152, pp.367-374, June.2019. Fig. 10. Computational results with spatial domain filters for Speckle noisy image p y g Fig. 11. Graph of evaluation parameters for removal of Speckle noise using various filters [5] N.Jyothi and D.Madhavi, " PSO Optimized Log Gabor QBIC System," International Journal of Innovative Technology and Exploring Engineering, vol. 8, no. 11, pp. 1650-1654, 2019. [6] D.Madhavi, N.Jyothi and MD.K.M.Chisti, "Composite method for Image Retrieval," International Journal of Engineering and Advanced Technology, vol. 9, Issue 3, Feb.2020. [7] Tania Sheikh et al., "A Comparative Study of Various Image Filtering Techniques for Removing Various Noisy Pixels in Aerial Image", International Journal of Signal Processing, Image Processing and Pattern Recognition, Vol. 9, no. 3, 2016 [8] W. Liu, J. Liu, L.Huang, Q. Du, and Y. Wang, "Performance analysis of reduced-dimension subspace signal filtering and detection in sample-starved environment", Journal of the Franklin Institute, Vol. 356, No. 1, pp. 629-653,2019. [9] M. Burger, Y. Korolev, C. Schönlieb, and S.Stollenwerk, "A total variation based regularizer promoting piecewise-Lipschitz [9] M. Burger, Y. Korolev, C. Schönlieb, and S.Stollenwerk, "A total variation based regularizer promoting piecewise-Lipschitz Fig. 11. Graph of evaluation parameters for removal of Speckle noise using various filters ISSN: 2395-1303 Page 24 http://www.ijetjournal.org reconstructions", arXiv preprint, Vol. 1903, No. 05079, 2019. [10] M. Salloum, K. Johnson, J. Bishop, J. Aytac, D.Dagel, and B. Van, "Adaptive wavelet compression of large additive manufacturing experimental and simulation datasets", Computational Mechanics, Vol. 63, No. 3, pp. 491-510, 2019. ISSN: 2395-1303 ISSN: 2395-1303 Page 25 http://www.ijetjournal.org
https://openalex.org/W2807381562	https://www.ssoar.info/ssoar/bitstream/document/75467/1/ssoar-jebo-2020-garz_et_al-Partisan_selective_engagement_evidence_from.pdf	English	null	Partisan selective engagement: Evidence from Facebook	Journal of economic behavior & organization	2,020	cc-by	20,495	www.ssoar.info www.ssoar.info Partisan selective engagement: evidence from Facebook Garz, Marcel; Sörensen, Jil; Stone, Daniel F. Veröffentlichungsversion / Published Version Zeitschriftenartikel / journal article Empfohlene Zitierung / Suggested Citation: Garz, M., Sörensen, J., & Stone, D. F. (2020). Partisan selective engagement: evidence from Facebook. Journal of Economic Behavior & Organization, 177, 91-108. https://doi.org/10.1016/j.jebo.2020.06.016 a r t i c l e i n f o Article history: Received 13 February 2020 Revised 14 June 2020 Accepted 15 June 2020 Available online 29 June 2020 JEL classiﬁcation: D83 D91 L82 Keywords: Filter bubble Media bias Political immunity Social media Polarization Article history: Received 13 February 2020 Revised 14 June 2020 Accepted 15 June 2020 Available online 29 June 2020 This study investigates the effects of variation in “congeniality” of news on Facebook user engagement (likes, shares, and comments). We compile an original data set of Facebook posts by 84 German news outlets on politicians that were investigated for criminal of- fenses from January 2012 to June 2017. We also construct an index of each outlet’s me- dia slant by comparing the language of the outlet with that of the main political parties, which allows us to measure the congeniality of the posts. We ﬁnd that user engagement with congenial posts is higher than with uncongenial ones, especially in terms of likes. The within-outlet, within-topic design allows us to infer that the greater engagement with congenial news is likely driven by psychological and social factors, rather than a desire for accurate or otherwise instrumental information. Keywords: Filter bubble Media bias Political immunity Social media Polarization © 2020 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license. ( http://creativecommons.org/licenses/by/4.0/ ) Marcel Garz a , ∗, Jil Sörensen b , Daniel F. Stone c a Jönköping International Business School, P.O Box 1026, SE-551 11 Jönköping, Sweden b Hamburg Media School, University of Hamburg, Germany c Bowdoin College, United States https://doi.org/10.1016/j.jebo.2020.06.016 0167-2681/© 2020 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license. ( http://creativecommons.org/licenses/by/4.0/ ) Partisan selective engagement: evidence from Facebook Garz, Marcel; Sörensen, Jil; Stone, Daniel F. Veröffentlichungsversion / Published Version Zeitschriftenartikel / journal article Empfohlene Zitierung / Suggested Citation: Garz, M., Sörensen, J., & Stone, D. F. (2020). Partisan selective engagement: evidence from Facebook. Journal of Economic Behavior & Organization, 177, 91-108. https://doi.org/10.1016/j.jebo.2020.06.016 Empfohlene Zitierung / Suggested Citation: Garz, M., Sörensen, J., & Stone, D. F. (2020). Partisan selective engagement: evidence from Facebook. Journal of Economic Behavior & Organization, 177, 91-108. https://doi.org/10.1016/j.jebo.2020.06.016 Empfohlene Zitierung / Suggested Citation: Garz, M., Sörensen, J., & Stone, D. F. (2020). Partisan selective engagement: evidence from Facebook. Journal of Economic Behavior & Organization, 177, 91-108. https://doi.org/10.1016/j.jebo.2020.06.016 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 Diese Version ist zitierbar unter / This version is citable under: https://nbn-resolving.org/urn:nbn:de:0168-ssoar-75467-7 Journal of Economic Behavior and Organization 177 (2020) 91–108 Contents lists available at ScienceDirect 1 We refer to news that is favorable to one’s preferred political party, or unfavorable to the ideologically distant party, as congenial, and to the opposite type of news as uncongenial. This binary notion of congeniality is useful for illustrative purposes and when describing the theoretical background in Section 2 . However, our empirical analysis accounts for continuous changes in the degree of congeniality. E-mail address: marcel.garz@ju.se (M. Garz). 1. Introduction In Section 2 , we present a highly stylized model of engagement with political news, to guide the interpretation of our results. We show that if social media users are motivated by the desire to provide Friends with information that is useful for either instrumental or intrinsic reasons, then users should in general be more likely to engage with uncongenial news as with congenial news. If users have other motivations—such as expressing how they feel, signaling their party loyalty or the validity of their political views, or wishing to persuade Friends to support one’s own preferred party—then users will be more likely to engage with congenial news. Thus, the model clariﬁes that selective engagement with congenial news is unlikely to be motivated by the desire to share useful information. While intuitive, this point stands in contrast to the substantial strand of the media bias theory literature showing that selective exposure to like-minded news can be driven by rational information-seeking behavior ( Gentzkow et al., 2015 ). We do not model the implications of engagement for the beliefs of a user’s Friends. But it is clear that if Friends are generally like-minded and do not fully account for a user’s psychological and strategic motives for engagement, then engaging more with congenial news will tend to push one’s Friends toward being more partisan, while engaging with uncongenial news equally or more than congenial news would have a moderating effect. In Section 3 we discuss the data and method for constructing a measure of ideological similarity between each outlet and party. We use the Facebook Graph Application Programming Interface (API) to download all 2,042,415 posts by these outlets, including information on the posts’ time of publication, content, type, and popularity. We then compare the lan- guage in these posts with the language used by the political parties in their election programs. Speciﬁcally, we compute the cosine distance between the text vectors for each of the 756 outlet-party combinations in our sample. This measure directly translates into a measure of congeniality in the context of our study: The larger the ideological distance, the more congenial the news about the lifting of immunity to an outlet’s average reader. In addition, we develop a search routine that is based on a combination of keywords and time parameters to identify posts about the cases of liftings of immunity. 1. Introduction It is well known that concerns about “echo chambers” and “ﬁlter bubbles”—citizens limiting their news consumption to belief-conﬁrming or amplifying sources—have grown rapidly in recent years. The corresponding academic literature has grown as well; see Guess et al. (2018) for a recent review. They conclude that “selective exposure to like-minded political news is less prevalent than you think.” Weak evidence for partisan selective exposure undermines the common suspicion that new media may be a primary cause of political polarization ( Sunstein, 2017 ; Bail et al., 2018 ). But exposure to news, and how one responds to news, are of course not the same thing. It is entirely possible that politically congenial news 1 is not only more likely to be believed by partisan media consumers, but that congeniality could ✩ We thank Maja Adena, Anna Kerkhof, Gregory Martin, Johannes Münster, Gaurav Sood, seminar participants in Hamburg, Jönköping, at the 2018 Le- uphana Workshop on Microeconomics, the 2018 Nordic Conference on Behavioral and Experimental Economics, the 2019 Economics of Media Bias Work- shop, the 2019 ZEW Conference on the Economics of ICT, and the 2019 CESifo Area Conference on Economics of Digitization for helpful comments and suggestions. We are also grateful to Katharina Brunner and Felix Ebert for sharing their extended results on Facebook users’ media preferences. Declara- tions of interest: none. This research did not receive any speciﬁc grant from funding agencies in the public, commercial, or not-for-proﬁt sectors. ∗Corresponding author. g j ( ) 1 We refer to news that is favorable to one’s preferred political party, or unfavorable to the ideologically distant party, as congenial, and to the opposite type of news as uncongenial. This binary notion of congeniality is useful for illustrative purposes and when describing the theoretical background in Section 2 . However, our empirical analysis accounts for continuous changes in the degree of congeniality. 92 M. Garz, J. Sörensen and D.F. Stone / Journal of Economic Behavior and Organization 177 (2020) 91–108 also lead to greater engagement with the news. Consumers may be more likely to endorse, pass on, respond to, or simply pay greater attention to more congenial news. These reactions could ultimately inﬂuence one’s own political beliefs and actions, and those of fellow citizens, with important welfare effects. , , p In this paper, we study such selective engagement (a term we propose to parallel the already standard term “selective exposure”). 1. Introduction We examine how Facebook users’ likes, shares, and comments on news posts vary depending on the political congeniality of the post. Selective engagement with congenial news would support those arguing that new media can be a signiﬁcant factor driving polarization (e.g., Lelkes et al., 2017 ; Gentzkow et al., 2018 ; Levy, 2019 ; Schwarz, 2019 ; Shmargad and Klar, 2020 ). In addition, it would likely suggest that social motives dominate information motives among users—an aspect emphasized by recent studies that link social media to hate crime ( Müller and Schwarz, 2018a ; 2018 b) and political protests ( Qin et al., 2017 ; Acemoglu et al., 2018 ; Enikolopov et al., 2019 ). Studying selective engagement is challenging because the nature of most news stories varies in many ways. Both the political congeniality of the story, and other aspects of the story that may affect consumer interest, such as the topic’s importance, vary across stories in ways that are typically diﬃcult to observe and measure. We address these challenges by studying variation in engagement with news stories on a ﬁxed topic: the lifting of the immunity of German politicians. Political immunity reduces the risk that members of parliament can be manipulated by the threat of arbitrary prosecution. The requirements to lift a politician’s immunity are strictly regulated, and immunity is only lifted when there is strong suspicion of involvement in criminal activity, such as tax evasion, embezzlement, or child pornography. In line with previous evidence, 2 we assume that the lifting of immunity for a politician in one’s preferred party is bad or uncongenial news, whereas it is good or congenial news when the immunity of an ideologically distant politician is lifted. We verify the validity of this assumption in Section 3.3 . Besides straightforward coding of congeniality, another key advantage of the topic of immunity being lifted is it that these events occur regularly, and are reported on frequently, by many outlets. During our period of investigation (January 2012 to June 2017), we observe 107 cases of German national and state representatives’ immunity being lifted. Our sample of Facebook news pages includes 84 outlets, of which several can be classiﬁed as mainstream pages with centrist coverage, as well as some at the left and right ends of the political spectrum. 2 A large body of literature suggests that politicians being involved in scandal regularly lose vote shares, refrain from running for reelection, or resign (e.g., Costas-Pérez et al., 2012 ; Hirano and Snyder, 2012 ; Larcinese and Sircar, 2017 ; Welch and Hibbing, 1997 ). The negative effects might be even stronger in our setting, as the act of lifting somebody’s political immunity requires hard evidence of criminal behavior, whereas political scandals may be simply based on unsubstantiated allegations. 1. Introduction Our estimation results imply that users do engage more with more congenial stories. Users like, share, and comment posts about liftings of immunity more often when the posts relate to politicians from the opposite political camp, compared to posts about ideologically similar representatives. According to the baseline speciﬁcation point estimate, an increase in congeniality by one standard deviation raises the average number of likes per post by 89%. The increases in shares and comments are 54% and 29%, respectively. Due to the within-outlet, within-topic research design of our study, these differ- ences can most likely be explained by the psychological and social factors mentioned above, and not due to demand for decision-relevant information or simply the desire to hold accurate beliefs (see Garz et al., 2020 ). An important potentially confounding factor is Facebook’s news feed algorithm, which is designed to maximize engage- ment by providing the content that users prefer. The algorithm could select those political immunity stories that users ﬁnd congenial (e.g., Claussen et al., 2019 ; Shmargad and Klar, 2020 ), and thus the greater engagement could be exclusively driven 93 M. Garz, J. Sörensen and D.F. Stone / Journal of Economic Behavior and Organization 177 (2020) 91–108 by the algorithmic exposure to congenial posts. If so, this would provide indirect evidence of partisan selective engagement. We also investigate this possibility by estimating our model with data from Twitter—when the platform did not have an algorithmically curated timeline—and obtain similar results. Thus it is unlikely that the greater engagement with congenial posts is completely driven by Facebook’s news feed algorithm. by the algorithmic exposure to congenial posts. If so, this would provide indirect evidence of partisan selective engagement. We also investigate this possibility by estimating our model with data from Twitter—when the platform did not have an algorithmically curated timeline—and obtain similar results. Thus it is unlikely that the greater engagement with congenial posts is completely driven by Facebook’s news feed algorithm. The interpretation of our main ﬁnding that users engage more with congenial posts for psychological and social reasons is only valid if the ideology of the outlets approximates the ideology of the users that engaged with the political immunity story posts in our sample. If this assumption is violated and the engagement with these posts is instead driven by users that do not share the outlets’ ideology, instrumental or intrinsic motives would likely dominate. 1. Introduction However, individual-level data on these users indicate that outlet and user ideologies are suﬃciently matched in our context. Furthermore, to complement the direct analysis of engagement, we compile a case-outlet panel and investigate the sup- ply of posts, and ﬁnd that supply is approximately unbiased. Thus the outlets do not cater to their readers by overreporting congenial cases, or such attempts are offset by differences in the costs of news production: Ideologically close outlets usu- ally have more background knowledge about and better connections to the politician in question, which makes it cheaper to produce news items and therefore easier to post uncongenial stories here. Our paper primarily contributes to the literature by shedding light on the mechanisms driving user engagement on social media. Bakshy et al. (2015) investigate, among other things, the distribution of average self-reported ideologies of users who share a given article. They ﬁnd that these averages are highly polarized: Most articles are shared by users who are either consistently liberal or conservative. This alone does not imply the sharing of congenial news, but they note other results suggesting this (that Fox News articles are shared by users who are mostly conservative, and Huﬃngton Post articles shared by mostly liberal users). An et al. (2014) obtain similar results, ﬁnding that self-reported partisans are more likely to share news from like-minded sources. Our paper conﬁrms these results, but differs in that we study multiple types of engagement and, perhaps more importantly, that we focus on news on just one topic. Fixing the topic allows us to distinguish between rational-information forces and psychological/social explanations for selective engagement with congenial news. Our paper complements Pogorelskiy and Shum (2018) , who study news sharing and voting on an artiﬁcial social network. Their ex- perimental set-up allows them to control for the true informational content of news especially cleanly. We conﬁrm their ﬁnding that subjects are more likely to share congenial news, but provide evidence based on observational, real-world data. In addition, we contribute to the literature on measuring media slant. Similar to the method proposed by Gentzkow and Shapiro (2010) , we compare the language of the most important parties in Germany with the language in the posts of the news outlets to construct an index of slant. 1. Introduction The bias of German media outlets has been investigated before; for instance, based on vocabulary and party mentions ( Dallmann et al., 2015 ), the tonality of reports ( Dewenter et al., 2019 ), and slant perceptions of readers ( Polisphere, 2017 ). In contrast to these studies, we do not restrict our investigation to a small number of national, leading media but provide a measure of slant for a more comprehensive set of outlets. For instance, our sample covers all types of news media (i.e., online, print, and broadcasting) and includes the most important regional outlets. More importantly, by calculating the ideological congruence between outlets and individual parties, our investigation is not limited to the position of media in the political left-right spectrum. That is, our slant index accounts for all sorts of ideological differences, such as progressive vs. conservative, egalitarian vs. elitist, authoritarian vs. libertarian, or religious vs. secular. Covering these dimensions is an important aspect when investigating multi-party systems. Finally, our ﬁndings contribute to the literature that investigates the role of media for political accountability, especially to research on news coverage about transgressions of politicians (e.g., Ferraz and Finan, 2008 ; Di Tella and Franceschelli, 2011 ; Puglisi and Snyder, 2011, 2015 ; Nyhan, 2014 ; Garz and Sörensen, 2019 ). In democratic societies, news media are con- sidered crucial watchdogs, allowing voters to make informed decisions. A central question in this literature is how well media transmit politically relevant information. We evaluate this aspect in a social media environment, by studying the supply of news about politicians that are investigated for criminal behavior. 2. Theory and empirical strategy We use the basic framework of Gentzkow et al. (2015) . The state of the world is binary, θ ∈ { L, R }, and news is binary as well, n ∈ { n L , n R }, with P r( n L \| L ) = P r( n R \| R ) = π > 0 . 5 . Thus n L ( n R ) is evidence that state L ( R ) is true ( Pr ( L \| n L ) > Pr ( L ), Pr ( R \| n R ) > Pr ( R )). The state could be interpreted as the relative valence of a politician ( θ = L would imply the leftist politi- cian has superior valence to the rightist). News about immunity for a politician is evidence about her/his relative valence. Facebook user i has prior p i = P r( θ = R ) , then exogenously observes the news, n , and then makes a decision about whether to engage with it on Facebook or not ( e i = 1 or 0). We examine several possibilities for the media consumer’s objective function to determine basic comparative statics to guide the empirical analysis and interpretation of results. The main goal is to clarify ideas, which is why we keep the model simple and the analysis largely informal. At the end of the section, we summarize the main results, explain how we can use the theory to interpret our empirical ﬁndings, and discuss potential differences between likes, shares, and comments. 2.1. Instrumental information 2.1. Instrumental information in which v ( X i , θ) is i ’s payoff from action X i in state θ, −i refers to the set of i ’s N Facebook Friends, X i ′ ( e i ) refers to the action of Friend i ′ given e i , and c > 0 is a per-user cost of engagement. That is, i cares about her own action given the state, and the actions of those in her network. The cost of engagement may be from an attention cost to others that i internalizes. The cost may also be due to i ’s concern about bothering Friends, losing credibility with Friends, or Friends paying less attention to i ’s engagements. There can also be a ﬁxed cost related to clicking the like/share button or writing a comment, without loss of generality. Assume, for simplicity, that i believes that i ’s Friends are only exposed to n due to i ’s engagement. This assumption is most appropriate for shares, but loosely applies to likes and comments, since these actions could attract attention to posts. If the action matches the state, then v ( X i , θ) = 1 , and v ( X i , θ) = 0 otherwise. It is then optimal for i to choose X L given n if Pr i ( L \| n ) > 0.5 and X R if Pr i ( R \| n ) > 0.5. 3 We say that n has instrumental value for i if n causes i to change her action from her ex ante optimal choice. 4 Thus, if p i > 0.5 and so X R is ex ante (prior to news) optimal, then n = n R has no instrumental value, and it is straightforward to show that n = n L has instrumental value if π > p i . Suppose all of i ’s Friends had the same prior. Then it is clear that v ( X i ′ ( e i ) , θ\| n ) is unaffected by whether e i = 0 or e i = 1 if n = n R since X i ′ = X R for all i ′ regardless. 2.1. Instrumental information Thus if n = n R , then e i = 1 imposes a cost and no beneﬁt, and so e i = 0 is i ’s optimal choice. If n = n L , then e i = 1 is optimal if ( 1 /N ) i ′ ∈−i [ E i (v ( X i ′ ( e i = 1 , θ\| n L )) ) −E i (v ( X i ′ ( e i = 0 , θ\| n L )) ) ] > c. This condition requires that the expected beneﬁt from providing instrumental information to a Friend (information that causes her optimal action to change) exceeds the cost of engagement, which is very plausible, so we assume that this condition holds. Suppose i ’s network is now heterogeneous. For simplicity, assume: p i ′ ∈ { p i , 1 −p i } . That is, some of i ’s Friends have priors favoring L symmetrically to those that favor R . Let f = P r( p i ′ = p i ) , i.e., the fraction of i ’s Friends who are like-minded. In this case, n R has instrumental value for i ’s left-leaning Friends, and n L has instrumental value for right-leaning Friends. Due to the symmetry assumption, this value is the same for any given individual. Let α denote this value for an individual, which is equal to E i (v ( X i ′ ( e i = 1 , θ\| n )) ) −E i (v ( X i ′ ( e i = 0 , θ\| n )) ) given that n has instrumental value for i ′ . Engaging with news creates the same cost for each Friend, and an average beneﬁt of f α if the news has instrumental value for right-leaning Friends, and an average beneﬁt of ( 1 −f ) α if the news has instrumental value for left-leaning Friends. Thus, writing the engagement choice as a function of the news realization, e i ( n L ) = 1 is now optimal if f α > c , i.e. f > c / α, and e i ( n R ) = 1 is optimal if ( 1 −f ) α > c, i.e., ( α −c ) /α > f. 3 All results, except those of Section 2.2 , are equivalent if consumers have heterogeneous tastes rather than priors. 4 The usage of the term here, referring to the realization of n , varies slightly from the more standard usagereferring to the value of n ex ante, but is similar in spirit. 5 There may even be some special cases for parameter values and model assumptions in which congenial news offered more instrumental value than uncongenial news. 2.1. Instrumental information First, suppose the consumer is only interested in instrumental information: information improving the quality of another decision. Let X i ∈ { X L , X R } denote the other decision. This decision could be something as simple as which candidate to M. Garz, J. Sörensen and D.F. Stone / Journal of Economic Behavior and Organization 177 (2020) 91–108 94 support in a public opinion poll. A politician’s standing in the polls could affect their “political capital” and consequently real policy outcomes. If the consumer or her Friends were perfectly indifferent between actions prior to news, the news would always provide instrumental information. We focus on other cases in which the consumer has one strictly preferred action prior to observing news; without loss of generality suppose this is X R . Speciﬁcally suppose i chooses ei given n to maximize the expectation of: (1) u i ( X i , X −i , e i , θ\| n ) = v ( X i , θ\| n ) + i ′ ∈−i v ( X i ′ ( e i ) , θ\| n ) −Nc e i , in which v ( X i , θ) is i ’s payoff from action X i in state θ, −i refers to the set of i ’s N Facebook Friends, X i ′ ( e i ) refers to the action of Friend i ′ given e i , and c > 0 is a per-user cost of engagement. That is, i cares about her own action given the state, and the actions of those in her network. The cost of engagement may be from an attention cost to others that i internalizes. The cost may also be due to i ’s concern about bothering Friends, losing credibility with Friends, or Friends paying less attention to i ’s engagements. There can also be a ﬁxed cost related to clicking the like/share button or writing a comment, without loss of generality. Assume, for simplicity, that i believes that i ’s Friends are only exposed to n due to i ’s engagement. This assumption is most appropriate for shares, but loosely applies to likes and comments, since these actions could attract attention to posts. 3 All results, except those of Section 2.2 , are equivalent if consumers have heterogeneous tastes rather than priors. 4 The usage of the term here, referring to the realization of n , varies slightly from the more standard usagereferring similar in spirit. 5 There may even be some special cases for parameter values and model assumptions in which congenial news offered more instrumental value than uncongenial news. 3 All results, except those of Section 2.2 , are equivalent if consumers have heterogeneous tastes rather than priors. 4 The usage of the term here, referring to the realization of n , varies slightly from the more standard usagereferring to the value of n ex ante, but is similar in spirit. 5 h b l f l d d l h h l ff d l l h 2.3. Psychological utility, persuasion, and signaling 2.3. Psychological utility, persuasion, and signaling Next, we consider several cases in which i ’s objective does not depend on the accuracy of i ’s Friends’ beliefs. The impli- cations for engagement in each of these cases are straightforward, so we omit mathematical analysis and simply discuss the effects that seem most plausible. First, assume that user i does not gain utility from news informing an action or accuracy of beliefs, but instead i ’s news utility increases when the news conﬁrms i ’s priors or supports an outcome that i hopes will occur, perhaps creating antic- ipation utility. Gentzkow et al. (2015) refer to this case as psychological utility. Clearly in this case i would be more likely to engage in the form of liking more congenial news, if i has any preference for the honest expression of this psychological utility, which is plausible ( Brennan and Hamlin, 1998 ; Abeler et al., 2016 ). Similarly, if i internalized the psychological utility of like-minded Friends, i would be more likely to engage with more congenial news to draw Friends’ attention to this news. Another reason for engaging with news unrelated to the desire for others to be well-informed about the state is that i may wish to use the news to persuade others to take a particular view about the state. If i is, for example, a committed rightist, i may wish to maximize the beliefs of others that θ = R independent of the realization of n . In this case, clearly it is optimal to share n = n R only. This case could easily be formalized by making u i a function of Friends’ beliefs that θ = R . This case would be even more relevant when i ’s Friends are more diverse or even left-leaning. This mechanism is perhaps also most relevant to news stories being shared. Another mechanism that may drive engagement orthogonal to the desire to provide useful information is signaling. One thing i may wish to signal to like-minded Friends in her network is that i is a strong partisan (to show loyalty, similarity, etc.). Choosing e i ( n R ) = 1 and e i ( n L ) = 0 would be an informative signal of gaining psychological utility from n = n R and sharing this for expressive value. 2.1. Instrumental information Stone / Journal of Economic Behavior and Organization 177 (2020) 91–108 95 the sign of the relationship between congeniality and engagement would be the same. In addition, for users with the same priors, the most natural equilibrium would still be symmetric. the sign of the relationship between congeniality and engagement would be the same. In addition, for users with the same priors, the most natural equilibrium would still be symmetric. 2.2. Intrinsic information 6 The difference between the prior and posterior after prior-conﬁrming news, Pr ( θ = R \| n R ) −p, is less than that after prior-opposing news, p − Pr ( θ = R \| n L ) , given p > 0.5. The latter difference minus the former reduces to p( 1 −p )( 2 π −1 )( 1 ( 1 −π) p+ π( 1 −p ) − 1 π p+( 1 −π)( 1 −p ) ) , which is unambiguously positive. 2.1. Instrumental information Ignoring knife-edge cases, there are now four possibilities: (1) i engages with all news ( ( α −c ) /α > f > c/α); (2) i engages with no news ( c/α > f > ( α −c ) /α); (3) i engages with n L only ( f > max { c/α, ( α −c ) /α} ); (4) i engages with n R only ( f < min { c/α, ( α −c ) /α} ). ) i engages with n L only ( f > max { c/α, ( α −c ) /α It is natural to think that f is greater than 0.5, given the prevalence of homophily (e.g., Bakshy et al., 2015 ; Halberstam and Knight, 2016 ). Making this assumption rules out case 4, since min { c/α, ( α −c ) /α} ≤0 . 5 . Thus, even allowing for het- erogeneous Friends, right-leaning consumers are not more likely to engage with n R rather than n L . Three additional points are worth noting before proceeding. First, if a user’s political preferences or beliefs are suﬃ- ciently strong, then uncongenial information would be less likely to have instrumental value for the user, but may still be instrumental for like-minded Friends with weaker preferences. Users might be less likely to share uncongenial information for strategic reasons in this case. Second, if the set of actions were larger, then it would be more likely that sharing conge- nial information with like-minded Friends would have instrumental value. For example, if the action set included a middle option (“to abstain”), and like-minded Friends prefer this option ex ante, then sharing congenial news would be more likely since this could persuade Friends to take the right-leaning action. 5 However, in general with richer action spaces, unconge- nial news would likely continue to have just as much, or more, instrumental value, for the same reason that this is true for the binary case (uncongenial information would be more likely to change a user’s optimal action). Third, if users take into account that Friends may see other Friends’ engagement for the same news, each user could be less likely to engage with any given post, knowing that other Friends may also engage with it. The equilibrium might change to mixed strategies, but M. Garz, J. Sörensen and D.F. 2.3. Psychological utility, persuasion, and signaling This case could be formalized by making i ’s type (“partisanship”, e.g., strength of prior favoring R ) uncertain, assuming that more partisan types are more interested in persuasion or the psychological utility of themselves or others, and that less partisan types get utility from a reputation term that increases in Friends’ beliefs of that type being more partisan. This case is more likely to be relevant when homophily is more common (when i ’s Friends are largely politically like-minded). An additional signaling possibility is the following. Suppose i may have processed some information inaccurately in ar- riving at i ’s current belief p i and i cares about her reputation with her Friends for processing information accurately. News of n R would be positive evidence in support of prior accuracy of interpretation by i , and n L would be negative evidence. Thus, again i would be more likely to engage with n R , and again this is easily formalized with a reputation term (this time, representing Friends’ beliefs about i ’s information processing ability, which would increase in consistency of new informa- tion with how i processed past information). This case is perhaps more likely to occur when i ’s Friends are diverse since non-like-minded Friends are more likely to need convincing that p i > 0.5 is a positive signal about i ’s ability. Both signal- ing cases are more likely for the more noticeable types of engagement, shares, and perhaps also comments, but could also explain “liking” engagement. 2.4. Connection between theory and empirical approach 2.2. Intrinsic information Another possibility is that the Facebook user’s objective is a function of information for intrinsic and not instrumental reasons. That is, it is possible that users want to hold beliefs that are as accurate as possible, and want their Friends to do so as well. The form of the objective function in Eq. (1) could still be applied in this case, with X i now denoting i ’s posterior probability that state R is true, and the states L and R correspond to θ taking values of 0 and 1, with v (.) denoting a loss function that increases in the distance between X i and θ. In this case, the beneﬁt of news, which is the impact on accuracy of beliefs, would be greater when the news conﬂicts with priors. This is because given a prior that favors R , beliefs change more after n L rather than n R . 6 Hence, again i would be more likely to engage with n L so long as a majority of Friends have like-minded priors favoring R . Thus engagement with uncongenial news ( n L ) seems equally or more likely than engagement with congenial news. Engagement with congenial and uncongenial news would be equally likely for this case if Facebook users were heterogeneous due to differences in tastes rather than differences in priors. (2) If engagement is motivated by other factors (expressive or anticipation utility, persuasion, or signaling), then users are more likely to engage with news that is more congenial. Thus, to link the theoretical considerations to the empirical analyses, we need to make the assumption that the outlet’s ideology is the same as the ideology of the users engaging with the political immunity story posts published by that outlet. Previous empirical evidence from US newspapers supports this as- sumption, showing that the average slant of a paper usually matches the ideology of the consumers in its area of circulation (e.g., Gentzkow and Shapiro, 2010 ; Puglisi and Snyder, 2015 ). How plausible is it that outlet and reader ideologies are matched in our context? It is arguably much easier for online users than newspaper readers to switch between outlets with different ideologies. Thus it is not suﬃcient to test if the ideology of the average reader matches that of the outlet, but we need to verify that those users who actually engaged with a political immunity story post share the ideology of the outlet publishing it. Similar to Bond and Messing (2015) , we examine these users’ like proﬁles to determine if they are “fans” of the Facebook pages of the political party involved in the post. We describe the procedure at the beginning of Appendix C in detail. We do not use the individual-level data in our main speciﬁcations because some users do not have their like proﬁles public, which may introduce selection bias. The data are nonetheless useful to compare the outlets’ average slant with the ideology of the relevant users. According to Fig. C1, users that engaged with a political immunity story post are more likely to be “fans” of the involved party if the outlet is on average slanted towards that party. Thus the ﬁgure suggests that the engagement with the posts in question primarily comes from users that share the ideology of the publishing outlet, which allows us to rule out that the second and fourth scenarios described in the previous paragraph play an important role here. With congruent ideologies, we are therefore able to use our main theoretical predications about individual motivations for engagement with (un-)congenial posts to interpret the empirical outlet-level results. Note that congeniality can be empirically measured in two ways, by looking at (a) bad news for an ideologically dis- tant party and (b) good news for a close party. (2) If engagement is motivated by other factors (expressive or anticipation utility, persuasion, or signaling), then users are more likely to engage with news that is more congenial. Both ideas refer to the relationship between user engagement and congeniality at the individual level , describing a situa- tion where the message of a post is in line with the views of an individual user. However, our empirical analyses in Section 4 relate to congeniality at the outlet level , evaluating if the news is compatible with the outlet’s ideology. That is, in our main estimating Eq. (7) , the dependent variable is the user engagement with the political immunity story post published by outlet n on case c , and the key explanatory variable is the ideological distance between outlet n and party p of the politician in question (and hence, the congeniality of that post). It is crucial to consider different scenarios when interpreting the empirical outlet-level results in light of the individual- level theory. A ﬁrst scenario is that the user engages with a post from a like-minded outlet followed by this user (i.e., a “subscribed” outlet that shares the user’s ideology). In this case, the ideological distance between outlet n and party p is a good proxy for the ideological distance between the user and the party involved in the political immunity story. Second, the user may also follow the Facebook page of an outlet with a different ideology. In this scenario, the outlet-level congeniality of the post does not capture the congeniality of that post for the user, because outlet and user ideologies are not matched. Third, the user engages with a post shared by a Friend or recommended by Facebook. If this post is from an outlet with a similar ideology as the user, the outlet-level measure of congeniality is again a good proxy for the congeniality of the post from the user’s perspective. Fourth, a post shared by a Friend or recommended by Facebook is from an outlet with a different ideology than the user, in which case congeniality at the outlet-level differs from the congeniality for that user. This fourth scenario could be particularly relevant if the user’s network of Friends is more heterogenous. Given that our empirical approach investigates congeniality at the outlet level, any results would be misleading if the second and/or fourth scenarios dominate in practice. (2) If engagement is motivated by other factors (expressive or anticipation utility, persuasion, or signaling), then users are more likely to engage with news that is more congenial. Higher engagement with either type of news is likely an indication that psychological and social factors matter, since both types are congenial to partisans. However, investigating good news for a close party would be less clean-cut, as motivations for intrinsic or instrumental information might also be at play here, at least in the context of a multi-party system. The reason is that ideological differences between parties are sometimes small (e.g., there have been political overlaps between the Greens (“Grüne”) and the Left Party (“Linke”), or between the Chris- tian (“CDU”) and Free Democrats (“FDP”)), and voters in multi-party systems often have a small set of close parties they consider. Thus engagement with good news for a close party could be an indication that users seek information to decide which party exactly to vote for. Focusing on political immunity stories avoids this kind of confounding, as these stories fall in the category of bad news for an ideologically distant party. 2.4. Connection between theory and empirical approach A summary of these results is as follows. Assuming that there is at least some homophily (more of a user’s Friends are politically like-minded than not): (1) If engagement is motivated by providing typically like-minded Friends with intrinsically or instrumentally useful infor- mation, social media users are in general more likely to engage with uncongenial news. M. Garz, J. Sörensen and D.F. Stone / Journal of Economic Behavior and Organization 177 (2020) 91–108 96 (2) If engagement is motivated by other factors (expressive or anticipation utility, persuasion, or signaling), then users are more likely to engage with news that is more congenial. 3.1. Politicians under criminal investigation In Germany, members of the national and state parliaments are generally protected from judicial and police measures. Unless apprehended while committing an offense, a representative can only be prosecuted or arrested if the parliament grants authorization. A few parliaments adopt lists of cases in which prosecution is possible without an explicit approval of the committee in charge; the authorities merely have to notify the parliament 48 h before taking action. However, in most cases, judicial or police measures have to be authorized. Usually, the act of liftings somebody’s immunity consists of two procedural steps. The parliamentary committee in charge ﬁrst issues a formal request, after which the ﬁnal decision on the lifting of immunity is made by parliament. Both steps might take place at the same day, but in many cases several days pass between the request and the decision. Lifting somebody’s immunity implies that the prosecution has suﬃcient evidence to initiate criminal proceedings. Some proceedings relate to minor transgressions (e.g., defamation, driving under the inﬂuence), whereas others pertain to severe felonies (e.g., child pornography). The prominence of the politicians varies as well, ranging from ordinary members of state parliaments to former Federal President Christian Wulff. In addition to the legal consequences that are associated with a lifting of immunity, such as indictment and conviction, public approval and intra-party support often decline. In some cases, politicians under investigation are excluded from their party or have to step down. Most information on cases of liftings of immunity come from oﬃcial parliamentary records. In a few cases, we com- plement missing data by publicly available information from other sources (e.g., press archives and search engines); see Garz and Sörensen (2017) for further details on collecting the data. Between January 2012 and June 2017, there are 107 cases pertaining to 80 politicians that belong to 9 different parties, after excluding six cases of politicians without party aﬃliation. 2.5. Types of engagement So far, we have talked about Facebook user engagement mostly in general terms, but likes, shares, and comments can be expected to vary in how strongly they respond to the congeniality of the content, if the engagement is motivated by expressive or anticipation utility, persuasion, or signaling. When a user likes a post, this action is visible below the post itself. There is a relatively low probability that Facebook will select the action of liking into Friends’ news feeds, unless they are followers of the publishing outlet. The cost of engagement is low, because liking is done with one click and only some Friends’ attention is drawn. 97 M. Garz, J. Sörensen and D.F. Stone / Journal of Economic Behavior and Organization 177 (2020) 91–108 Sharing a post implies that the content will be reposted on the user’s timeline. The likelihood that Facebook selects this action into Friends’ news feeds is comparatively high. Thus the cost of engagement is higher than in the case of likes, even though sharing also requires just one click. Sharing a post implies that the content will be reposted on the user’s timeline. The likelihood that Facebook selects this action into Friends’ news feeds is comparatively high. Thus the cost of engagement is higher than in the case of likes, even though sharing also requires just one click. Commenting is an action that becomes visible below a post and also has a relatively high probability of appearing in Friends’ news feeds. This type of engagement certainly has the highest cost of engagement, because the user needs to write something rather than just click a button, in addition to the internalized attention costs of Friends. Comments are used to add information to a post, and since this information could be either critical or conﬁrmatory, this behavior could occur whether the post is congenial or not. If engagement is motivated by social or psychological factors, we therefore expect liking to be most strongly associated with the congeniality of a post. The relationship between congeniality and sharing is presumably less strong, due to the higher cost of engagement. Commenting involves the highest cost and might not necessarily be used to conﬁrm a post message, which is why we expect the weakest and perhaps most ambiguous relationship here. 3. Data Our data cover the time from January 2012 to June 2017. Before 2012, we do not observe much activity on the Facebook pages of the outlets in our sample. In fact, many outlets did not have an oﬃcial Facebook page before 2012. Our period of investigation ends in June 2017, shortly before we started collecting the data. 3.1. Politicians under criminal investigation 3.3. Retrieval of political immunity story posts We are interested in retrieving those posts that address an imminent, requested, or realized lifting of immunity. It is not diﬃcult to ﬁnd many of these posts because there are no synonyms for the German word “Immunität”. In addition, the term is always used as a noun or compound noun (e.g., “Immunitätsaufhebung”) and there are no word corruptions. Thus, the term “Immunität” (truncated at the end) is a very sharp and effective keyword in this context. We focus on the post message, which has a similar function as the headline or sub header of a traditional news article. Journalists include buz- zwords in the post message to signal its topic to the reader. The underlying news article very likely addresses the lifting of somebody’s immunity if the word immunity is included in this message. 8 In addition, there are posts that address the lifting of somebody’s immunity without explicitly using the word “Immunität”. Such posts are usually published around the date of the request or decision to lift the immunity, discussing the intentions of the prosecution to open criminal proceedings, the particularities of the transgression, and potential or actual consequences of the case. It is also straightforward to retrieve these kinds of posts by searching for the name of the politician in question and comparing the date of the post with the date of the lifting of immunity. To not omit many true positives, we do not initially restrict the search to posts that contain both the ﬁrst name and the last name, because the media often only uses the last name of the politician. An exception are German last names that are very common (e.g., Beck, Müller) or are also used as other words, such as “Mächtig” (power- ful) or “Junge” (boy), which is when the media usually uses the full name to avoid misunderstandings. In these cases, we retrieve only those posts that contain the ﬁrst name and the last name. Based on these considerations, we develop a simple routine to search the downloaded page data for posts that are likely about liftings of immunity. 3.2. Facebook news pages and posts Newspapers, news magazines, and newscasts nowadays provide access to their content online, often complementing their traditional ways of distributing information. Similar to pure Internet news portals, these outlets have Facebook representa- tions which they use to post content. A Facebook post is an individual entry in a page’s feed. In addition to its message, a post may include a photo, a video, or a link to external content. News outlets are usually interested in getting Facebook users exposed to and engage with their posts. User engagement refers to liking, sharing, and commenting the post, as well as clicking on external links. In the case of news pages, these links usually redirect the user to full articles on the outlets’ websites. Our selection of outlets is guided by audience reach and the goal to fully cover the political left-right spectrum. To con- struct a sample that fulﬁlls these criteria, we include all media outlets that focus on general or politics news, have an oﬃcial Facebook page, and are listed in at least one of the following rankings: (a) the 75 most visited news websites according to the German audit bureau of circulation (Informationsgesellschaft zur Feststellung der Verbreitung von Werbeträgern, IVW ), (b) the 75 most popular outlets on Facebook and Twitter according to the social media monitoring project 10,0 0 0 Flies ( http://www.10 0 0 0ﬂies.de/ ), or (c) the Süddeutsche ranking of the most-liked Facebook pages by politically interested users ( https://bit.ly/2COYrAV ). The IVW is one of Germany’s most established provider of audience reach data. Its ranking is based on the number of page impressions of news websites in June 2017. Thus, it does not speciﬁcally reﬂect the popularity of outlets on Facebook but more generally online. The 10,0 0 0 Flies ranking also refers to June 2017, but it is based on the num- ber of likes, reactions, shares and comments on Facebook, as well as the number of likes and retweets on Twitter, generated by websites with their content. The ranking by Süddeutsche is the most speciﬁc one to the context of this study. Referring 98 M. Garz, J. Sörensen and D.F. 3.2. Facebook news pages and posts Stone / Journal of Economic Behavior and Organization 177 (2020) 91–108 to the time from October 2016 to March 2017, their ranking lists the 100 Facebook pages most often liked by politically in- terested Facebook users; it consists of a separate list for each of the six most popular German parties these users also like. Based on these criteria, our sample consists of 84 Facebook news pages (many outlets appear in all three rankings). The selection includes Facebook representations of all national newspapers, all national news magazines, the most important re- gional newspapers, the most important national newscasts, and the largest online news outlets. The sample also comprises known left- and right-wing outlets, such as Compact-Magazin, Junge Freiheit, Junge Welt, PI-News , and taz (Die Tageszeitung). See Table A1 for a full list of outlets. We use the Facebook Graph API to download the entire content of these pages, including all 2,042,415 posts. The number of posts varies across outlets, with a minimum of 2154 ( Monitor ) and a maximum of 92,955 ( N24 ). Given these quantities, we have a suﬃciently large sample to accurately estimate each outlet’s ideology, even for those outlets that were less active on Facebook. For each post, we record the date and time of publication, the message text, the type (i.e., link, status, photo, video, or event), the link to the underlying news article, the link to a possible picture, as well as the number of likes, shares, and comments. 7 We download the data in the second half of August 2017—at least six weeks after the most recent posts—to guarantee that we register the “ﬁnal” engagement measures (99.9% of user engagement takes place in the 15 days after a post is published; see Lee et al., 2018 ). 8 We also check other related search terms, including synonyms and word corruptions, such as “Staatsanwalt” (prosecutor), “Ermittlung” (investigation), and “Strafverfahren” (criminal procedure), but using these terms barely leads to the retrieval of true positives, while substantially increasing the number of false positives. 7 In 2016, Facebook equipped the like button with further options to react to content, such as “love”, “wow”, and “angry”. Because of their novelty, we do not consider these reactions in our analyses. 3.3. Retrieval of political immunity story posts The routine identiﬁes all posts that (a) contain the name of the politician in question and the German word for immunity (truncated at the end) in their message text, article link, or picture link, or (b) only include the name but are published within seven days before and after the date of the request or decision to lift this politician’s immunity. This search procedure retrieves 1291 posts. Manually removing a few false positives—which are mostly due to homonymy—decreases this number to 1115 posts. Fig. A1 in Online Appendix A shows the distribution of these posts across outlets. Five outlets never posted about a political immunity story ( Lausitzer Rundschau, MAZ, Neue Westfälische, Südwest Presse , and WDR ), whereas the television news channel N24 had a record number of 88 posts. Importantly, about half of the outlets in our sample had more than 10 posts, which implies that we can estimate the relationship between the congeniality of news and user engagement based on a large number of diverse outlets. Table 1 , Panel B, provides further summary statistics. On average, a post receives about 84.1 likes, 16.9 shares, and 56.4 comments. Most of the posts simply include a link (81.2%), followed by status updates (11.0%). Few posts include a photo (6.9%) and even less a video (0.8%). We assume that the content of the retrieved posts is bad news for the accused politician. Allegations of criminal be- havior are usually associated with losses in vote shares (e.g., Welch and Hibbing, 1997 ; Costas-Pérez et al., 2012 ; Hirano and Snyder Jr., 2012 ). In some cases, such accusations cause politicians to refrain from running for reelection or to resign prematurely ( Garz and Sörensen, 2017 ; Larcinese and Sircar, 2017 ). The language used in the political immunity story posts reﬂects the detrimental implications of the act. Applying Rauh’s (2018) sentiment dictionary for German political language suggests that these posts are disproportionately negative. On average, the post messages contain 1.09 positively but 2.05 negatively connotated terms. Reports about a lifting of immunity always provide “actual news”, even if the public has heard rumors about the criminal behavior before. The act of lifting somebody’s political immunity implies that the prosecution has 99 M. Garz, J. Sörensen and D.F. Stone / Journal of Economic Behavior and Organization 177 (2020) 91–108 Table 1 Summary of the data. Mean SD Min. Max. 3.3. Retrieval of political immunity story posts Panel A: Case-level variables (N = 8988 ) Ideological distance ( dist ) 0.805 0.193 0.000 1.000 Posts -count 0.124 1.109 0.000 36.000 -count, excluding multiple-topic posts 0.120 1.075 0.000 36.000 -count, excluding multiple-politician posts 0.107 0.907 0.000 30.000 Panel B: Post-level variables (N = 1115 ) Ideological distance ( dist ) 0.624 0.193 0.000 0.998 Engagement (amount) -likes 84.079 294.562 0.000 6912.000 -shares 16.935 46.198 0.000 623.000 -comments 56.375 106.375 0.000 1862.000 Type of post (share) -link 0.812 0.391 0.000 1.000 -photo 0.069 0.254 0.000 1.000 -status 0.110 0.313 0.000 1.000 -video 0.008 0.090 0.000 1.000 Party aﬃliation of politician (share) -AfD 0.061 0.239 0.000 1.000 -CDU 0.273 0.446 0.000 1.000 -CSU 0.104 0.305 0.000 1.000 -Grüne 0.050 0.219 0.000 1.000 -Linke 0.065 0.246 0.000 1.000 -NPD 0.001 0.030 0.000 1.000 -Piraten 0.002 0.042 0.000 1.000 -SPD 0.445 0.497 0.000 1.000 collected hard evidence that is likely suﬃcient for a conviction (about 50% of these cases result in a guilty verdict; see Garz and Sörensen, 2017 ). Thus it is plausible to assume that political immunity stories are negative publicity for the politician in question, and by extension, for the politician’s party and supporters. collected hard evidence that is likely suﬃcient for a conviction (about 50% of these cases result in a guilty verdict; see Garz and Sörensen, 2017 ). Thus it is plausible to assume that political immunity stories are negative publicity for the politician in question, and by extension, for the politician’s party and supporters. However, it is conceivable that the outlets could attempt to defend the (criminal) behavior of an ideologically close politician, especially when the transgression is politically motivated. As a consequence, readers of these outlets might not perceive such posts as uncongenial. We manually identify all posts that defend the accused politician and verify that these posts do not pose a problem to our approach of measuring congeniality (see robustness checks in Online Appendices B and C). Speciﬁcally, we tag all posts that clearly (a) solidarize with the accused, (b) deplore the behavior of prosecuting authorities and parliamentary committees, or (c) contest the meaningfulness of prevailing law. 9 We also consider but do not pursue the option to tag these posts by using an automated approach. On the one hand, we are not aware of an automated procedure that could accurately evaluate these criteria. 9 We do not explicitly consider the linked news article, because its content likely correlates with the content of the post message. In addition, in most cases users engage with a post without reading the underlying story ( Gabielkov et al., 2016 ). 10 Our measure of media slant is exclusively based on the outlets’ Facebook posts for two reasons. First, our approach to measure congeniality of political immunity story posts on Facebook requires a measure of slant on Facebook. Other forms of news output by the outlets (e.g., their print versions, broadcasts, or websites) could by characterized by a different slant, since audiences on Facebook and outside likely differ. Second, there would be data availability issues for a large fraction of the outlets in our sample, as the entirety of the news output outside of Facebook often cannot be accessed. 3.3. Retrieval of political immunity story posts It is possible to measure the sentiment of the posts, as mentioned in the previous paragraph, but it would be a stretch to assume that a post would defend the accused politician if there was a positive sentiment. On the other hand, the number of posts that need to be evaluated is small enough for a manual approach to be feasible. There are 69 posts that meet at least one of the above-mentioned criteria (ca. 6.2% of all retrieved posts). In most cases, these posts pertain to left-wing and Green politicians that were accused of violating the right of free assembly, either by participating in anti-Nazi demonstrations or anti-nuclear movements. 3.4.1. Similarity between outlets and parties 3.4.1. Similarity between outlets and parties We use an approach similar to that proposed by Gentzkow and Shapiro (2010) to construct our measure of media slant. Speciﬁcally, we compare the language in the outlets’ Facebook posts 10 with the language used by the parties in their elec- tion programs. Before an election, German parties usually publish a document that provides details on their goals for the upcoming legislative session. These documents are a central element of the campaigns and thus widely discussed in the public. They circulate under the label election program or, in some cases, election platform, party program, party manifesto, or government program. We use all programs pertaining to the two national elections during our period of investigation (2013 and 2017), as well as the available state-level programs in that time. 100 M. Garz, J. Sörensen and D.F. Stone / Journal of Economic Behavior and Organization 177 (2020) 91–108 The election programs are used to identify characteristic terms that are typically used by the parties. With an average of 295,971 words per party, the documents provide a suﬃcient amount of text. We clean the texts (i.e., lower case trans- formation, word stemming, as well as removal of punctuation, numbers, stop words, formatting, and party references) and generate a matrix representation of the processed terms. We evaluate the importance of these terms by computing the prod- uct of the relative term frequency and the inverse document frequency (TF-IDF), which is a standard statistic in information retrieval for this kind of task (e.g., Jurafsky and Martin, 2008 ). Simply put, the TF-IDF is largest for terms that are often used by one but not the other parties; i.e., terms that appear frequently and uniquely in the parties’ election programs. The exact formula is in the notes to Table A2; the table itself shows each party’s 20 highest ranked terms to illustrate the approach. For instance, characteristic terms of the Left Party (Linke) are “erwerbslos” (unemployed), “superreich” (super-rich), and “neoliberal” (neoliberal), whereas typical terms by the right-wing party NPD are “Vaterland” (fatherland), “Massenzuwan- derung” (mass immigration), and “Ausländerkriminalität” (crime committed by foreigners). The Greens (Grüne) emphasize “Kohleausstieg” (fossil fuel phase-out), “Klimakrise” (climate crisis), and “Atomausstieg” (nuclear phase-out); and character- istic terms of the market-liberal FDP are “Vertragsfreiheit” (freedom of contract), “Schulfreiheitsgesetz” (autonomy education act), and “Träume” (dreams). 3.4.1. Similarity between outlets and parties These examples also show that it is not necessary to compute the TF-IDF for two- or three- word phrases because of the common usage of compound nouns in the German language. Next, we concatenate the 2,042,415 Facebook posts by the outlets in our sample and clean the message texts in the same way as the election programs. We compare the language of the outlets with the language of the parties by computing the cosine similarity (cp. Jurafsky and Martin, 2008 ) between the cleaned post messages and the most characteristic terms of the parties: si m n,p = T t=1 x t ,nyt ,p T t=1 x t, n 2 T t=1 y t, p 2 (2) Based on terms t , we compute this statistic for each of the 756 outlet-party combinations. The term vector x represents the post messages of outlet n , whereas y captures the language used by party p . Since we are interested in those terms that are highly characteristic for the language of each party, we focus on terms with particularly high TF-IDF values. Speciﬁcally, we only use the top 0.1% of each party’s characteristic terms, according to the TF-IDF statistic. Selecting this cut-off is arbitrary and subject to a trade-off. 11 Using only few party-speciﬁc terms increases the chances that we treat posts as neutral even if they are actually biased, as we might exclude terms that are indicative of a party’s ideology. An overly generous cut-off decreases the differences between the parties, because it involves including terms that are not often used, that are simultaneously used by the other parties, or both. Inspection of the retained terms suggests that the top 0.1% of the distribution of TF-IDF values balances the trade-off well. This cut-off selects between 48 (AfD, NPD) and 86 (FDP, SPD) terms per party, or a total of 582 expressions. These differences between parties reﬂect the uniqueness and length of the election programs. Thus parties with a broader range of unique topics have a greater weight when constructing the slant index. We plot levels of slant by outlet and party in Figs. A2 and A3. The graphs indicate that the outlets generally vary in their tendency to use ideologically relevant terms. For instance, we observe the lowest tendency to use terms for the regional outlet Badische Zeitung (cosine similarity cumulated over parties = 0.0034). 11 An alternative strategy would be to evaluate the outlets’ use of all terms included in the election programs, while applying the TF-IDF values as weights of “keyness”. Unfortunately, this strategy is computationally not feasible, given the size of the Facebook corpus. Tables B1, B2, C1, and C2 present robustness checks when using higher and lower cut-offs. 12 We also explore the possibility to measure slant at the level of individual posts, in an attempt to capture congeniality in a particularly detailed way. We discard this possibility because there would be endogeneity problems, as the slant of individual posts might directly affect user engagement. Measuring congeniality at the outlet level minimizes these problems. In addition, it would be practically infeasible to analyze post-level slant, because over 90% of the posts do not contain any ideologically relevant terms. Thus there would be hardly any variation to analyze, considering that our sample consists of 1115 political immunity story posts. 3.4.2. Score in the political left-right spectrum To illustrate the outcome of the computations, we rank the outlets in the political left-right spectrum. For that purpose, we use left-right scores of Germany’s political parties provided by the Manifesto Project ( Volkens et al., 2019 ). These scores are based on content analyses of the parties’ 2013 and 2017 election programs, following a standardized procedure that allows comparisons over time and across countries. As shown in Fig. 1 , the Pirate Party (Piraten), the Greens (Grüne), the Social Democratic Party (SPD), and the Left Party (Linke) can be found left of the mean score, whereas the Union parties (CDU/CSU), the Free Democratic Party (FDP), and the newly founded Alternative for Germany (AfD) are right of the center. 13 ( / ) y ( ) y y ( ) g Next, we regress the cosine similarity between outlet n and party p on a constant and outlet and party ﬁxed effects μn and θ p : si m n,p = a + μn + θp + ε n,p (4) The posts of some outlets are generally more similar to the parties’ election programs than the posts of other outlets; and the election programs of some parties are generally closer to the outlets’ posts than those of other parties. Using the residuals ɛ n, p from Eq. (4) accounts for these differences. Our measure of an outlet’s position in the left-right spectrum is the sum of the outlet’s similarity residuals ɛ n, p weighted by the parties’ score p on the left-right scale (as shown in Fig. 1 ): scor e n = P p=1 ε n,p scor e p (5) By construction, the outlets’ left-right score n is bounded between -1 and 1. Negative values suggest that an outlet’s post messages are slanted towards the parties left of the population mean, whereas positive values indicate slant towards the right side of the spectrum. By construction, the outlets’ left-right score n is bounded between -1 and 1. Negative values suggest that an outlet’s post messages are slanted towards the parties left of the population mean, whereas positive values indicate slant towards the right side of the spectrum. In Fig. 2 , we compare the resulting left-right score of the national outlets with the popularity of their Facebook pages. The distribution of these outlets resembles a bell curve. 3.4.1. Similarity between outlets and parties The socio-critical blog Nachdenkseiten , for which we measure the highest cumulated cosine similarity (0.0399) exceeds this value by a factor of 10. However, there are also differences in the extent of bias towards individual parties. For example, we observe outlets devoting between 4.1% ( Os- thessen News ) and 23.4% ( Zeit ) of their overall bias to the green party (Grüne), and between 0% (multiple outlets) and 10.9% ( Compact-Magazin ) to the far-right NPD. In the regressions, our measure of congeniality is the cosine distance between an outlet and a party (i.e., dis t n,p = 1 −si m n,p ). 12 To ease the interpretation of the results, we rescale the cosine similarity using its empirical minimum and maximum before computing the distance: dis t n,p = 1 − si m n,p −min ( sim ) max ( sim ) −min ( sim ) dis t n,p = 1 − si m n,p −min ( sim ) max ( sim ) −min ( sim ) dis t n,p = 1 − si m n,p −min ( sim ) max ( sim ) −min ( sim ) (3) The resulting measure varies between 0 and 1, with 1 reﬂecting the greatest observed ideological distance between an outlet and a party, and 0 indicating the greatest similarity. The empirical distribution of this distance measure (cp. Fig. A4) implies that there is much variation in the congeniality of political immunity story posts, which is useful when estimating the effects on user engagement. M. Garz, J. Sörensen and D.F. Stone / Journal of Economic Behavior and Organization 177 (2020) 91–108 101 101 Fig. 1. Left-right scores of political parties. Notes: The ﬁgure shows left-right scores of Germany’s main political parties, based on data from the Manifesto Project ( Volkens et al., 2019 ). The Manifesto Project derived theses scores by analyzing the content of the parties’ 2013 and 2017 election programs. More negative values indicate stronger leanings to the left, whereas more positive values imply stronger leanings to the right. For comparison, the Democratic and Republican parties in the US scored −20.58 and 32.97, respectively, on the same Manifesto Project scale (pertaining to the 2016 US presidential elections). For a recent journalistic comparison of German and US political parties see Chase (2017) . Fig. 1. Left-right scores of political parties. Fig. 1. Left-right scores of political parties. 13 Note that the outlets’ left-right scores are calculated without accounting for the right-wing NPD. The Manifesto Project data do include information about this party, given its low vote shares in recent elections (1.3% in 2013 and 1.0% in 2017). To verify that omitting the NPD does not distort our ranking, we compute another version of score n by replacing the Manifesto Project scores with survey-based left-right values collected by the 2016 Politbarometer surveys ( N = 17,556). The data capture respondents’ perceptions of the parties’ positions in left-right spectrum, including the NPD. Figs. A5 and A6 show the resulting ranking of outlets, which does not substantially differ from the baseline approach. However, the Politbarometer data have the disadvantage that survey responses can be subject to social desirability bias, which is why we relegate these ﬁgures to the appendix. Comparing our left-right score with existing measures of slant further conﬁrms our approach. There is a bivariate correlation of 0.80 when we compare our left-right score with the index of perceived slant by Polisphere (2017); see Fig. A7. 14 The distinction between national and regional outlets is based on the outlets’ self-description. Almost all regional outlets in our sample include a reference to some locality in their name. They differ from national outlets in that they offer local news on top of national and foreign news. Traditionally, printed copies of these outlets could be purchased at the newsstands in a certain area only, or reception of their terrestrial transmission was limited to some region. 3.4.1. Similarity between outlets and parties g g p p Notes: The ﬁgure shows left-right scores of Germany’s main political parties, based on data from the Manifesto Project ( Volkens et al., 2019 ). The Manifesto Project derived theses scores by analyzing the content of the parties’ 2013 and 2017 election programs. More negative values indicate stronger leanings to the left, whereas more positive values imply stronger leanings to the right. For comparison, the Democratic and Republican parties in the US scored −20.58 and 32.97, respectively, on the same Manifesto Project scale (pertaining to the 2016 US presidential elections). For a recent journalistic comparison of German and US political parties see Chase (2017) . 13 Note that the outlets’ left-right scores are calculated without accounting for the right-wing NPD. The Manifesto Project data do include information about this party, given its low vote shares in recent elections (1.3% in 2013 and 1.0% in 2017). To verify that omitting the NPD does not distort our ranking, we compute another version of score n by replacing the Manifesto Project scores with survey-based left-right values collected by the 2016 Politbarometer surveys ( N = 17,556). The data capture respondents’ perceptions of the parties’ positions in left-right spectrum, including the NPD. Figs. A5 and A6 show the resulting ranking of outlets, which does not substantially differ from the baseline approach. However, the Politbarometer data have the disadvantage that survey responses can be subject to social desirability bias, which is why we relegate these ﬁgures to the appendix. Comparing our left-right score with existing measures of slant further conﬁrms our approach. There is a bivariate correlation of 0.80 when we compare our left-right score with the index of perceived slant by Polisphere (2017); see Fig. A7. 14 The distinction between national and regional outlets is based on the outlets’ self-description. Almost all regional outlets in our sample include a reference to some locality in their name. They differ from national outlets in that they offer local news on top of national and foreign news. Traditionally, printed copies of these outlets could be purchased at the newsstands in a certain area only, or reception of their terrestrial transmission was limited to some region. 3.4.2. Score in the political left-right spectrum The score on the x axis is computed by mapping language similarities between outlets and parties to left-right scores of the parties provided by the Manifesto Project (see Section 3.4 for details). main explanatory variable—the ideological distance shown in Eq. (3) —has very similar means and standard deviations when comparing national and regional media. Thus we use variation in ideology from both types of outlets when estimating the relationship between congeniality and user engagement. It might seem puzzling that Facebook pages belonging to the same brand (i.e., Spiegel and Spiegel Online , as well as Zeit and Zeit Online ) have slightly different left-right scores. Using different pages operated by different social media editors is most likely part of product differentiation strategies that allow media companies to target different audiences (e.g., Anand et al., 2007 ; Gal-Or et al., 2012 ). Thus it is not implausible for these outlets to have different left-right scores. Overall, the positions of the outlets in the left-right spectrum shown in Figs. 2 and 3 concur with their reputations. For example, we obtain large positive scores for the right-wing outlets Junge Freiheit, PI-News , and Compact , whereas the social- ist newspaper Neues Deutschland , the socio-critical blog Nachdenkseiten , and the cooperative-owned taz (Die Tageszeitung) exhibit large negative scores. 3.4.2. Score in the political left-right spectrum The graph suggests that the most popular outlets—such as Bild, Spiegel Online , and Tagesschau —are fairly balanced; i.e., have left-right scores close to zero. Outlets at the left and right ends of the spectrum are less popular on Facebook. We do not ﬁnd such a pattern when looking at the regional outlets in Fig. 3. 14 This is plausible because regional outlets usually cater to the views of consumers in local news markets ( Gentzkow and Shapiro, 2010 ). These markets vary in size, which is a main factor of the regional outlets’ popularity on Facebook. In addition, the newspaper versions of the regional outlets are often local monopolists; as such they have incentives to cater to a broad ideological spectrum rather than a single ideology ( Gentzkow et al., 2015 ). The regional outlets also have a smaller range of left-right scores, which implies that their Facebook pages are ideologically more balanced than the national ones, as can be expected in locally concentrated news markets. Note, however, that we compute and display the left-right scores only for illustrative purposes, mainly to validate that our approach of measuring ideology yields plausible results. Our 102 102 M. Garz, J. Sörensen and D.F. Stone / Journal of Economic Behavior and Organization 177 (2020) 91 108 Fig. 2. Popularity and slant of Facebook news pages, national outlets. Notes: The page likes refer to August 2017. The score on the x axis is computed by mapping language similarities between outlets and parties to left-right scores of the parties provided by the Manifesto Project (see Section 3.4 for details). main explanatory variable—the ideological distance shown in Eq. (3) —has very similar means and standard deviations when comparing national and regional media Thus we use variation in ideology from both types of outlets when estimating the Fig. 2. Popularity and slant of Facebook news pages, national outlets. Notes: The page likes refer to August 2017. The score on the x axis is computed by mapping language similarities between outlets and parties to left-right scores of the parties provided by the Manifesto Project (see Section 3.4 for details). Fig. 2. Popularity and slant of Facebook news pages, national outlets. Notes: The page likes refer to August 2017. M. Garz, J. Sörensen and D.F. Stone / Journal of Economic Behavior and Organization 177 (2020) 91–108 To begin, we investigate the supply of posts, given the availability of news material. For that purpose, we estimate the effect of the congeniality of the case on the number of posts on a given case, by a given outlet: post s n,c = b 1 + b 2 dis t n,p + μn + ϕ c + ε n,c 4.1. Supply of posts To begin, we investigate the supply of posts, given the availability of news material. For that purpose, we estimate the effect of the congeniality of the case on the number of posts on a given case, by a given outlet: (6) post s n,c = b 1 + b 2 dis t n,p + μn + ϕ c + ε n,c M. Garz, J. Sörensen and D.F. Stone / Journal of Economic Behavior and Organization 177 (2020) 91–108 103 Fig. 3. Popularity and slant of Facebook news pages, regional outlets. Notes: The page likes refer to August 2017. The score on the x axis is computed by mapping language similarities between outlets and parties to left-right scores of the parties provided by the Manifesto Project (see Section 3.4 for details). Fig. 3. Popularity and slant of Facebook news pages, regional outlets. Notes: The page likes refer to August 2017. The score on the x axis is computed by mapping language similarities between outlets and parties to left-right scores of the parties provided by the Manifesto Project (see Section 3.4 for details). where dist is the cosine distance between outlet n and party p of the politician involved in case c , as calculated in Eq. (3) . Note that the congeniality of the case derives from the ideological congruence between the outlet and the party aﬃliation of the politician in question: Cases in which an outlet is slanted towards the party of the politician are uncongenial, because the lifting of immunity is bad news for the average reader of this outlet. In contrast, cases are congenial if an outlet is ideologically different from the party of the politician. Thus, high values of dist reﬂect congenial constellations, whereas small values indicate uncongenial cases. We estimate this effect conditional on outlet and case ﬁxed effects. The outlet ﬁxed effect μn captures unobserved differences across outlets; for example, due to popularity, social media strategy, or aﬃnity for the topic. The case ﬁxed effect ϕc accounts for the particularities of the case, such as the point of time of the lifting of immunity, the severity of the transgression, the party of the suspect, or the reputation of the politician, which might all result in differences in news value. We compute two-way clustered standard errors by outlet and case. 4.1. Supply of posts (1) (2) (3) # Posts # Posts, excluding multiple-topic posts # Posts, excluding multiple-politician posts Ideological distance −0.412 −0.393 −0.296 (0.342) (0.327) (0.234) R 2 0.283 0.278 0.263 Notes: N = 8988 (107 cases, 84 outlets). OLS estimates. The column headers state the dependent variables. All models include outlet and case ﬁxed effects. Standard errors (in parentheses) are clustered by outlet and case. ∗p < 0.10, ∗∗p < 0.05, ∗∗∗p < 0.01. parameters problem. In fact, maximum likelihood estimates often fail to converge with the data at hand, because there are many outlet-case combinations without any variation in the amount of posts. parameters problem. In fact, maximum likelihood estimates often fail to converge with the data at hand, because there are many outlet-case combinations without any variation in the amount of posts. Results are summarized in Table 2 . All speciﬁcations indicate a negative relationship between the ideological distance and the supply of posts. Thus the number of posts slightly decreases when the congeniality of the case to the outlet’s average reader increases. However, the estimated coeﬃcients are statistically insigniﬁcant and imply very small effect sizes. For example, the coeﬃcient of −0.412 in Column ( 1 ) indicates that a one standard deviation increase in distance (0.193) decreases the number of posts by 0.079. This decrease corresponds to approximately 7.1% of the standard deviation of the amount of posts. p Fig. B1 supports the interpretation that the congeniality of the case does not affect the number of posts related to that case. To rule out that this ﬁnding is caused by our speciﬁc approach of capturing congeniality, we evaluate the robustness of the estimates when we construct the distance measure in a different way. We obtain similar results when using alternative TF-IDF cut-offs to construct this measure (Tables B1 and B2) and when we use a measure of congeniality based on the Polisphere (2017) index of slant (Table B3). As discussed in Section 3.3 , a small fraction of posts defends the accused. These posts might have a different congeniality, but our estimates do not substantially differ when we distinguish between these posts and “ordinary” political immunity stories (Table B4). To rule out that our results are driven by outliers, we exclude the most active outlets (Table B5) and drop the most prominent cases (Table B6). 4.1. Supply of posts where dist is the cosine distance between outlet n and party p of the politician involved in case c , as calculated in Eq. (3) . Note that the congeniality of the case derives from the ideological congruence between the outlet and the party aﬃliation of the politician in question: Cases in which an outlet is slanted towards the party of the politician are uncongenial, because the lifting of immunity is bad news for the average reader of this outlet. In contrast, cases are congenial if an outlet is ideologically different from the party of the politician. Thus, high values of dist reﬂect congenial constellations, whereas small values indicate uncongenial cases. We estimate this effect conditional on outlet and case ﬁxed effects. The outlet ﬁxed effect μn captures unobserved differences across outlets; for example, due to popularity, social media strategy, or aﬃnity for the topic. The case ﬁxed effect ϕc accounts for the particularities of the case, such as the point of time of the lifting of immunity, the severity of the transgression, the party of the suspect, or the reputation of the politician, which might all result in differences in news value. We compute two-way clustered standard errors by outlet and case. We use different versions of the dependent variable when estimating Eq. (6) . The basic version simply counts all posts per outlet and case. In the second version, we exclude posts that refer to multiple issues (i.e., posts also addressing other topics than the lifting of somebody’s immunity). For instance, the Facebook pages of newscasts sometimes use a post to list the main topics of their upcoming show. The third version excludes posts related to multiple politicians from different parties. The outlets sometimes report on multiple politicians simultaneously, because the cases occur at the same time or to compare a case with a previous lifting of immunity. It would be optimal to use estimation procedures that account for the distributional characteristics of these count vari- ables, such as Poisson or negative binomial models. We use OLS to estimate Eq. (6) though, because maximum likeli- hood estimators are biased when modeling panel data with (two-way) ﬁxed effects; see Greene (2004) on the incidental M. Garz, J. Sörensen and D.F. Stone / Journal of Economic Behavior and Organization 177 (2020) 91–108 104 Table 2 Supply of political immunity story posts and congeniality of cases. 15 It would be possible to analyze the sentiment of the comments related to the political immunity story posts. However, we do not believe that content analyses would be particularly informative in the context of our study, because negative or positive sentiment does not necessarily correspond to the congeniality of a post. For example, if users express that they are sad when the immunity of a politician from their preferred political camp is lifted, those comments would be characterized by a negative sentiment. If instead the immunity of an ideologically distant politician is lifted, users might express anger, which would also be registered as negative sentiment. Thus the sentiment of the comments is unlikely to reﬂect a post’s congeniality. 4.1. Supply of posts Excluding outlets and cases with zero posts does not affect the results either (Table B7). Another concern is that OLS estimates are biased when the dependent variables are count variables. However, we obtain qualitatively similar results when we estimate linear probability models with binary dependent variables that indicate whether the outlet posted about a case or not (Table B8). Another possibility is that only certain outlets adjust their supply of posts to the congeniality of the case, but we do not ﬁnd any differences between left- and right-leaning or between centered and more extreme outlets (Table B9). It is also conceivable that outlets do not adjust the number of posts but change certain qualitative elements. For instance, outlets could be more inclined to include a call to action in a post when the case is more congenial. This is not the case though (Table B10). In addition, there is no evidence that the congeniality of the case affects the characteristics of the posts, such as the length of the post message, the usage of photos and videos, and the share of posts published on Sundays or at night (Table B11). In conclusion, there is no evidence that outlets cater to the preferences of users by posting more about more congenial cases. Absence of evidence is not necessarily evidence of absence though. It is possible that attempts by outlets to satisfy consumer demand for congenial posts are offset by other factors. For instance, cost advantages could make it attractive to post about less congenial cases: Outlets that are ideologically close to a party likely have more background knowledge about and better connections to politicians from that party, which implies lower costs of producing news items about them. In contrast, it might be more diﬃcult for outlets to obtain certain information when politicians from ideologically distant parties are involved. Such cost differences inﬂuence the supply of news items, which in turn affects the activity of outlets on Facebook. 4.2. User engagement 4.2.1. Estimation and results Table 3 shows the results of estimating Eq. (7) . In general, the estimates do not substantially differ for the models with (Columns 4 to 6) and without (Columns 1 to 3) control variables. Throughout, the coeﬃcient of the distance variable has a positive sign. Thus the estimates suggest that users engage more with posts, the greater the level of congeniality of the posts. Assuming that outlet and user ideologies are approximately matched (cp. Fig. C1), the positive relationship implies that the users’ engagement is likely motivated by psychological and social factors, as discussed in Section 2 . According to Columns ( 4 ) to ( 6 ), a one standard deviation increase in distance (0.193) raises user engagement by approximately 74.4 likes, 9.2 shares, and 16.4 comments. In relative terms, likes, shares, and comments increase by 88.5%, 54.3%, and 29.1%, respec- tively. The coeﬃcients are signiﬁcant at the 5% and 10% level in the case of likes and shares, and insigniﬁcant for comments. Both the size of the coeﬃcients and their signiﬁcance levels are compatible with the theoretical expectations formulated in Section 2.5 . That is, we observe the strongest association for likes, which involve the lowest costs of engagement. The re- lationship is slightly weaker for shares, presumably because of the somewhat higher engagement cost. The low correlation between comments and engagement can likely be explained by the relatively high engagement cost and the fact that com- ments are not necessarily used to endorse the content. Graphical evidence conﬁrms the positive relationship between the congeniality of the posts and user engagement (see Fig. C2). A series of robustness checks suggests that our results are not substantially affected by speciﬁc choices made when constructing our measure of congeniality. Speciﬁcally, we obtain qualitatively similar results when we use alternative TF- IDF cut-offs to select ideologically relevant expressions (Tables C1 and C2), construct a measure of congeniality based on differences in the left-right spectrum (Table C3), or create a distance measure based on the Polisphere 2017 ranking of outlets (Table C4). We evaluate if posts that defend the accused affect the results, which is not the case (Table C5). We also verify that our results are not driven by outliers in the engagement variables (Fig. C3), particularly prominent cases (Table C6), or two outlets with extreme left-right scores (Table C7). 4.2.1. Estimation and results The control variables include the type of the post, the length of the post message, the outlets’ monthly average number of likes over all published posts, day of the week and hour of the day ﬁxed effects, the overall and the outlet-speciﬁc number of previous posts on the same case, the number of days since the ﬁrst post on the same case, a dummy to capture posts that refer to multiple politicians of different parties, and a dummy to capture posts about multiple topics. Standard errors (in parentheses) are clustered by outlet and case. ∗p < 0.10, ∗∗p < 0.05, ∗∗∗p < 0.01. μn and ϕc , account for unobserved outlet- and case-speciﬁc characteristics. The variable vector X includes controls for the type of the post (i.e., link, photo, video, status, and event), the length of the post message (number of characters), and a dummy variable to capture posts that refer to multiple politicians of different parties. Another binary variable captures posts that cover multiple topics because user reactions to such posts do not necessarily refer to the lifting of somebody’s immunity. In addition, dummy variable sets account for the hour of the day and the day of the week, because the timing of publication likely affects user engagement as well. We also include the number of days since the ﬁrst post on a case, as well as the overall and the outlet-speciﬁc number of previous posts on the same case to capture variation due to potential effects of the news cycle. Finally, we include the outlets’ average monthly number of likes over all published posts to account for differences in popularity over time and across outlets. Again, we cluster standard errors by outlet and case. Here the effective numbers of outlets and cases are 79 and 49, respectively: Some outlets never posted about a political immunity story, and various cases did not receive any posts. Technically, it would be possible to include month and year ﬁxed effects, as well as a time trend polynomial to account for general, time-related patterns. However, such patterns are almost entirely absorbed by the case ﬁxed effects because most posts are published at the time of the lifting of immunity. Including these variables does not affect the results but leads to extremely large variance inﬂation factors due to multicollinearity, which is why we prefer to omit the additional controls. 4.2.1. Estimation and results 4.2.1. Estimation and results We estimate versions of the following model to explore the effect of the congeniality of posts on users: estimate versions of the following model to explore the effect of the congeniality of posts on users (7) engagemen t i,n,c = c 1 + c 2 dis t n,p,c + c 3 X i,n,c + μn + ϕ c + ε i,n,c engagemen t i,n,c = c 1 + c 2 dis t n,p,c + c 3 X i,n,c + μn + ϕ c + ε i,n,c engagemen t i,n,c = c 1 + c 2 dis t n,p,c + c 3 X i,n,c + μn + ϕ c + ε i,n,c in which we use each of our three measures of user engagement (likes, shares, and comments) related to post i , published by outlet n on case c as left-hand side variables. 15 Again, dist captures the ideological distance between the outlet and party p of the politician in question (and hence, the congeniality of a story on lifting immunity). The outlet and case ﬁxed effects, M. Garz, J. Sörensen and D.F. Stone / Journal of Economic Behavior and Organization 177 (2020) 91–108 105 Table 3 User engagement and congeniality of posts. (1) (2) (3) (4) (5) (6) Likes Shares Comments Likes Shares Comments Ideological distance 434.1 ∗ 56.53 ∗ 96.90 387.8 ∗∗ 48.15 ∗ 85.51 (237.9) (33.55) (66.36) (195.4) (28.28) (53.25) Controls No No No Yes Yes Yes R 2 0.269 0.485 0.494 0.333 0.520 0.526 Notes: N = 1115. OLS estimates. The column headers denote the dependent variables. All models include outlet and case ﬁxed effects. The control variables include the type of the post, the length of the post message, the outlets’ monthly average number of likes over all published posts, day of the week and hour of the day ﬁxed effects, the overall and the outlet-speciﬁc number of previous posts on the same case, the number of days since the ﬁrst post on the same case, a dummy to capture posts that refer to multiple politicians of different parties, and a dummy to capture posts about multiple topics. Standard errors (in parentheses) are clustered by outlet and case. ∗p < 0.10, ∗∗p < 0.05, ∗∗∗p < 0.01. Table 3 Notes: N = 1115. OLS estimates. The column headers denote the dependent variables. All models include outlet and case ﬁxed effects. 4.2.2. Alternative explanations The positive relationship between the congeniality of posts and user engagement could be subject to reverse causality if ideologically motivated social media editors exploit certain features of Facebook to advocate their own or the outlet’s political agenda. There are several ways in which outlets can promote individual posts to increase the chances that these posts appear in users’ news feeds, which in turn increases the chances of engagement. Outlets can pay Facebook to “boost” posts, they can pay click farms to inﬂuence organic exposure, they can use social bots, they can pay inﬂuencers to spread the word (e.g., celebrities), and they can slant message texts. Editors could systematically promote those posts that support the outlet’s ideological goals and disregard the posts that contradict these goals. Speciﬁcally, outlets could use these tools to sway audiences with a different ideology. We cannot conclusively rule out this kind of behavior, but various pieces of evidence contradict the hypothesis that outlets attempt to persuade users on a large and successful scale. First, our ﬁnding that the supply of posts is approximately unbiased does not support this kind of behavior. Second, as discussed in Appendix B, we do not ﬁnd that the outlets manipulate the salience of posts in congenial cases by changing post characteristics (i.e., length, duration of coverage, and use of photos/videos) and the timing of publication (i.e., weekdays vs. weekends, night vs. day). If the actions of the editors were driven by ideological goals, we should already observe a bias at this stage. Third, persuasion would imply that outlets target partisan or uncommitted users with content that is not congenial to them, in which case we would not observe a positive relationship between congeniality and user engagement in the data. While not impossible, it is therefore unlikely that our results are driven by an ideologically motivated promotion of posts. It is also conceivable that other actors could attempt to persuade audiences by using bots, fake accounts, or trolling. This kind of inﬂuence is mostly known in the context of elections and politically motivated disinformation campaigns. We cannot rule out that political immunity stories are used to manipulate Facebook audiences. However, the posts in our sample often relate to low-level politicians and are published outside of election times, so it is unclear if there is enough at stake for disinformation campaigns to inﬂuence our results at a large scale. 4.2.2. Alternative explanations The robustness checks that exclude the most prominent cases offer some reassurance here (cp. Table C6). In addition, bots and trolls tend to share content from dubious sources, but the vast majority of posts in our sample were published by legacy media. Our heterogeneity analyses indicate that the relationship between congeniality and user engagement is mostly driven by centered outlets (cp. Table C9, Columns 4 to 6), which also suggests that political bots are unlikely to be a big factor here. Another alternative explanation relates to Facebook’s news feed algorithm, which predominantly selects congenial con- tent into users’ news feeds. The algorithm likely predicts the aﬃnity of users to certain outlets, parties, and politicians, as well as preferences for news categories (e.g., politics vs. sports) and topics within news categories (e.g., posts about trans- gressions of politicians vs. posts about policy making). It might also be possible that the algorithm distinguishes between congenial and uncongenial news within the same outlet in the speciﬁc context of liftings of immunity. However, as Bakshy et al. (2015) show, the algorithmic selection has a smaller effect on exposure to congenial content than individuals’ choices. To evaluate the role of the algorithm, we collect data on engagement with political immunity stories on Twitter (see Ap- pendix C for details). In contrast to Facebook, Twitter did not use an algorithm to expose users to tweets until January 2016. The platform only began to test an algorithmically curated timeline in February 2016. Before that, tweets were simply shown in reverse chronological order to users. 16 As Table C10 shows, Twitter users did engage more with more congenial political immunity stories prior to February 2016, despite the lack of algorithmic exposure. However, we also ﬁnd that the liking of congenial tweets has increased afterwards, whereas we do not observe signiﬁcant changes for retweets and replies. Thus the algorithmic content selection might amplify user engagement with congenial content, but it is unlikely that our Facebook results are exclusively driven by the news feed algorithm. Unfortunately, we cannot quantify how much of the effect estimated in Eq. (7) is driven by the news feed, since the Facebook and Twitter algorithms differ in certain details. A related possibility is that social media editors exploit tools (e.g., paid reach, inﬂuencers) to raise the chances that users are exposed to congenial content. 16 See https://blog.twitter.com/oﬃcial/en _ us/a/2016/never- miss- important- tweets- from- people- you- follow.html . 4.2.1. Estimation and results The user engagement could be driven by posts including a call to action (e.g., “What do you think about the accusations?”), but removing these posts does not change the results either (Table C8). Overall, the robustness checks conﬁrm the results of the baseline speciﬁcation, but the level of the robustness varies over the different engagement measures. We ﬁnd the most robust effects for likes. The evidence is slightly less robust for shares, and most speciﬁcations do not indicate signiﬁcant effects for comments. Again, this pattern matches the theoretical predictions discussed in Section 2.5 . p As discussed in Section 2 , the ﬁndings can be best explained by psychological and social factors. It “feels good” to en- counter congenial news, whereas it “feels bad” to receive uncongenial information. The instrumental information concept M. Garz, J. Sörensen and D.F. Stone / Journal of Economic Behavior and Organization 177 (2020) 91–108 106 used in many theoretical models of demand-driven media bias is not compatible with the result that consumers prefer congenial information within the same outlet. If consumers were behaving rationally and were seeking information with instrumental value, we should ﬁnd either (a) no within-outlet relationship between congeniality and user engagement or (b) users to be more engaging with uncongenial posts. This is not the case though. The absence of such a ﬁnding does not necessarily imply that the effect it not present, but it could mean that psychological and social factors are dominant. 4.2.2. Alternative explanations Outlets could steer different posts to different audiences, depending on the content. For instance, an outlet might explicitly target right-leaning users with political immunity stories about left-wing politicians, whereas left-leaning users could be targeted with posts about right-wing politicians. This kind of strategy would aim to maximize engagement by catering to user preferences. As with Facebook’s news feed algorithm, editor-driven exposure could amplify the positive relationship between congeniality and user engagement. Similarly, outlets could take direct measures to increase engagement metrics (e.g., social bots, engagement baiting). Checking the political immunity story posts in our sample, we ﬁnd that a small fraction of posts (4.8%) include calls to action (e.g., “What do you think about this?”). Excluding these posts does not change our results though (Table C8). We also check if there are other forms of engagement baiting (e.g., “Tag a friend that…”, “Like this if you think that…”, “Share with 10 friends if you…”) but do not ﬁnd any in our sample of political immunity story posts. M. Garz, J. Sörensen and D.F. Stone / Journal of Economic Behavior and Organization 177 (2020) 91–108 107 M. Garz, J. Sörensen and D.F. Stone / Journal of Economic Behavior and Organization 177 (2020) 91–108 5. Conclusion We investigate the effect of variation in congeniality in Facebook posts on user engagement. Using data on posts about the lifting of politicians’ immunity allows us to distinguish between messages that conﬁrm and contradict the beliefs of the average readers of 84 German news outlets on Facebook. Our results indicate that the supply of posts is approximately unbiased, whereas we ﬁnd systematic differences in user engagement. Posts with congenial messages receive substantially more likes than uncongenial messages. To lesser degree, this also applies to shares, but not necessarily to comments. These differences across the engagement metrics match our theoretical predictions. g g p It would be optimal to explicitly account for potential supply-side factors that affect users’ exposure to posts, such as ideologically motivated social media editors or Facebook’s news feed algorithm. However, it is unlikely for such factors to be large confounders in our within-outlet, within-topic approach, because exposure only translates into engagement if user preferences are compatible. Another limitation of our research design is the speciﬁc kind of news that we investigate. It is unclear whether our ﬁndings can be generalized to other contexts than transgressions of politicians. Regardless, the results have important theoretical implications. We show that psychological and social factors play a major role in shaping user engagement. However, many models of demand-driven media bias do not account for such factors (e.g., Burke, 2008 ; Chan and Suen, 2008 ; Sobbrio, 2014 ; Oliveros and Várdy, 2015 ; Fang, 2016 ). This is problematic because these models lead to different conclusions about the welfare implications of media bias than models that do account for psychological factors (e.g., Mullainathan and Shleifer, 2005 ; Gentzkow and Shapiro, 2006 ; Bernhardt et al., 2008 ; Stone, 2011 ): If preferences for congenial news are driven by the desire to hold accurate or otherwise instrumental information, the proliferation of ideologically diverse media is relatively likely to cause consumers to be better informed. If preferences for congenial news are based on psychological and social factors, as our paper suggests, concerns about ﬁlter bubbles leading to biased beliefs are more justiﬁed. Abeler, J. , Nosenzo, D. , Raymond, C. , 2016. Preferences for truth-telling. Econometrica 87, 1115–1153 . 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https://openalex.org/W3037295422	https://jecs.pl/index.php/jecs/article/download/1139/1003	Kinyarwanda	null	Cyberbullying prevention and intervention programs - are they enough to reduce the number of online aggressions?	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https://openalex.org/W2772035340	https://www.rasi.vr.uff.br/index.php/rasi/article/download/172/49	English	null	Beneficial Effects of Slow Steaming in Bulk Freight Markets	Revista de Administração, Sociedade e Inovação	2,017	cc-by	5,244	Emissions Reduction in Shipping Beyond Slow Steaming Taliese LaVerne (TAMUG) - tlaverne@tamug.edu Sarah Boone (TAMUG) Joan Mileski (TAMUG) - mileskij@tamug.edu Emissions Reduction in Shipping Beyond Slow Steaming Taliese LaVerne (TAMUG) - tlaverne@tamug.edu Sarah Boone (TAMUG) Joan Mileski (TAMUG) - mileskij@tamug.edu Emissions Reduction in Shipping Beyond Slow Steaming Taliese LaVerne (TAMUG) - tlaverne@tamug.edu Sarah Boone (TAMUG) Joan Mileski (TAMUG) - mileskij@tamug.edu ABSTRACT: Slow steaming has recently been adopted into normal practice by many maritime shipping companies for the fuel and monetary savings it offers. The practice also offers savings in Greenhouse Gas (GHG) emissions. With regulations coming into play such as the 2020 sulfur cap, slow steaming may be the least costly option for some maritime companies to adjust their operations. While some have accepted the new practice, there are still companies and vessels that see this exercise as a loss of revenue due to the extra time it takes to deliver goods to their destination. This paper reviews how rating ships by their GHG emissions per nautical mile can be directly related to factors other than slow steaming such as age, deadweight tonnage and cargo type. We propose that ships with poor ratings (E, F, G) should rely on slow steaming but may also improve their CO2 output addressing the other factors. Those with superior ratings (A, B, C, D) may also benefit from the implementation of slow steaming, but may also gain from practices addressing age, tonnage and cargo type. Further we will also examine how lowering emissions can benefit maritime businesses both economically and environmentally. Finally this paper reviews possible chain reactions that may occur if these eco-friendly shipping practices are observed. KEYWORDS: slow steaming, greenhouse gases, vessels, ecological transport 1. Introduction Mitigating Greenhouse Gas emissions has been a hot topic for the shipping industry in recent years. Without out a true way to measure emissions globally for shipping, the regulators such as the IMO preform estimations from previous years. In the Third IMO Study, it was concluded that “International shipping emissions for 2012 are estimated to be 796 million tonnes CO2 and 816 million tonnes CO2e for GHGs combining CO2, CH4 and N2O. International shipping accounts for approximately 2.2% and 2.1% of global CO2 and GHG emissions on a CO2 equivalent (CO2e) basis, respectively” (IMO, 2014). The question then becomes, how can the shipping industry further reduce its impact on the environment while still maintaining a profitable business? One operational option is slow steaming. Through the implementation of slow steaming, many benefits have been observed. By utilizing this practice, a ship will experience less wear and tear on the engine of the vessel due to lower speeds. This leads to less maintenance thus monetary savings can be seen. Fuel savings will also result from this operational adjustment. Reducing fuel usage will not only be monetarily beneficial but also environmentally as well. Burning less fuel contributes directly to GHG mitigation. Ports could also see benefits from slow steaming. The idea of scheduling a berth time after participating in slow steaming would be an incentive which could lead to mitigating congestion thus further alleviating emissions. This practice would mean less idling time outside of the port for the vessel participating in slow steaming. Some vessels idle for days waiting to get into port, constantly burning fuel. This idling contributes to higher operating costs, it also lends to further GHG emissions. By reducing congestion outside of ports by this berth scheduling incentive, multifaceted savings would be seen. Other factors that could influence vessel emissions are: age of the vessel, weight in deadweight tonnage (DWT), and cargo. Age of vessels while it may seem obvious, contributes to the amount to emissions produced. Deadweight tonnage again appear to have an evident correlation with emissions. Cargo carried has rarely been looked at as a factor but could offer insight to emissions. Emissions data from RightShip addresses these factors. This study could help define more ways for a company to save rather than to just slow down. 1. Introduction Ports could further utilize emissions data from organizations like RightShip, to provide incentives for ships higher rated vessels which could have a snowball effect for other ships to improve their practices and boost their ratings to receive those incentives. Another proposition in utilizing data like this would be for ships rated poorly rated ships, finding more stringent regulation targeted at them to improve their practices and decrease their CO2 emissions. RESUMO: O vapor lento foi recentemente adotado na prática normal por muitas companhias marítimas para a economia de combustível e monetária que oferece. A prática também oferece economias em emissões de gases de efeito estufa (GEE). Com os regulamentos que entram em jogo, como o limite de enxofre de 2020, o vapor lento pode ser a opção menos dispendiosa para algumas empresas marítimas ajustar suas operações. Enquanto alguns aceitaram a nova prática, ainda existem empresas e embarcações que vêem esse exercício como uma perda de receita devido ao tempo extra que leva para entregar bens ao seu destino. Este artigo analisa como a classificação dos navios por suas emissões de GEE por milha náutica pode ser diretamente relacionada a fatores como idade, tonelagem de peso morto e carga. Nós propomos que os navios com classificações ruins (E, F, G) encontrem regulamentos obrigatórios para diminuir o vapor ou melhorar sua produção de CO2 de alguma forma. Aqueles com classificações superiores (A, B, C, D) se beneficiariam de pacotes de incentivos ligados à sua implementação de vapor lento ou outras práticas. Também examinará como a redução das emissões pode beneficiar as empresas marítimas de forma econômica e ambiental. Finalmente, este artigo analisa possíveis reações em cadeia que podem ocorrer se essas práticas de transporte ecológico forem observadas. PALAVRAS-CHAVE: vapor lento, gases de efeito estufa, embarcações, transporte ecológico R. Desembargador Ellis Hermydio Figueira, 783, Bloco A, sl. 218, Aterrado. 27213-415 - Volta Redonda, RJ – Brasil www.uff.br Copyright © 2017 RASI. Todos os direitos, até mesmo de tradução, são reservados. É permitido citar parte de artigos sem autorização prévia, desde que seja identificada a fonte Recebido em 20/09/2017 Aceito em 08/11/2017 LaVerne, Boone & Mileski \|176 10.20401/rasi.3.2.172 Emissions Reduction in Shipping Beyond Slow Steaming 2. Literature Review Literature regarding the benefits of slow steaming for both cost and environmental benefits is becoming increasingly popular. Most of the research on this topic has concentrated on ship speed optimization and the benefits stemming from finding the optimal operating speed of the vessel. Between increased awareness of environmental issues, poor market conditions, and volatile fuel prices, there is more pressure than ever to find the most optimal speed to navigate these circumstances. http://www.rasi.uff.br http://www.rasi.uff.br RASI, Volta Redonda/RJ, v. 3, n. 2, pp. 175-187, jul./dez. 2017. LaVerne, Boone & Mileski \|177 10.20401/rasi.3.2.172 Fuel consumption by a vessel can vary based on a few different factors. Whoever is paying for fuel must make the most optimal decision possible. “Contracts many times refer to ‘utmost dispatch’ that encourage carriers to pursue speeds as fast as reasonably possible.” (Alvarez et al, 2010). Hydrodynamic characteristics also play a factor in fuel comsumption. Psaraaftis & Kontovas state, “hull condition can also be an important factor that influences the frictional resistance of a ship, and, as a result, fuel consumption” and continues with“ship resistance and hence fuel consumption at a given speed can be drastically different if the ship is full, empty or at an intermediate leading condition” (Psaraftis & Kontovas, 2014). Along with load of ship, fuel prices themselves are a critical piece to the optimal speed puzzle as well. During the financial crisis of 2009 there was a sharp rise in slow steaming due to the high cost of bunker fuel (Wong et al, 2015).“Fuel prices are a very critical determinant of fuel costs, and, as such, of the speed chosen by the vessel. In fact fuel price is the one of two main factors that play a critical role in the determination of ship speed (the state of the market being the other one)” (Psaraftis & Kontovas, 2014). Freight rates can have an impact on the decision of how fast or slow a ship chooses to go as well. Ultimately the decision of how quickly to arrive in port is associated with net profits. If there is money to be made or saved by arriving to the port as soon as possible shippers and charterers will forgo the environmentally friend options such as slow steaming. 2. Literature Review However, through further literature review, there are findings which show that there are more reasons than just a depressed market where ships and the environment can benefit from reduced speed. So far, slow steaming has been “a response to depressed market conditions and/or high fuel prices” (Psaraftis & Kontovas, 2014). However, there is a notable relationship between the environment and speed of a vessel which is gaining some much-deserved attention in literature. As a ship’s speed increases, it will burn more fuel thereby emitting more pollution. With higher demands from consumers, shippers feel the pressure to respond and by supplying goods equal to demand which creates even more congestion in ports. Between the ship’s increased speed to meet demand and the idling in a queue to get into congested ports, unnecessary fuel is being used, therefore producing an even greater amount of emissions. “International shipping accounts for approximately 2% of global carbon emissions” (Mander, 2016). Pierre Cariou’s paper, Is slow steaming a sustainable means of reducing CO2 emissions from container shipping? shows that “Reducing a vessel’s speed by 10% decreases emissions by at least 10-15%” proving that “one positive effect of slow steaming is that it lowers CO2 emissions that are proportional to the amount of fuel burned” (Cariou, 2011). In another study done by Ching-Chin Chang and Chia-Hong Chang, they used an activity-based method to evaluate fuel consumption against CO2 emissions and found that “speed reductions of 10%, 20%, and 30% reduced fuel consumption by 27.1%, 48.8%, and 60.3% and CO2 emissions by 19%, 36%, and 51%, respectively” (Chang & Chang, 2013). An increasing amount of studies are linking speed reduction as a viable way to mitigate carbon emissions and other greenhouse gases. It is clear that slow steaming provides benefits in air emissions but there is also a relationship between the speed of a vessel and monetary benefits found from decreasing speed. “Reducing speed could also have important side benefits: cost reduction is one, and helping a depressed market in which shipping overcapacity is the norm these days is another” (Psaraftis & Kontovas, 2012). Market conditions which include factors such as fuel prices and freight rates can also be compared versus a ships speed. RASI, Volta Redonda/RJ, v. 3, n. 2, pp. 175-187, jul./dez. 2017. 2. Literature Review In fact, “NYK Group investigated the cost-benefit analysis and emission reduction of slow steaming, indicating a slow steaming of reducing the speed of a vessel with 8,000 Twenty-foot Equivalent Unit (TEU) containers from http://www.rasi.uff.br RASI, Volta Redonda/RJ, v. 3, n. 2, pp. 175-187, jul./dez. 2017. LaVerne, Boone & Mileski \|178 10.20401/rasi.3.2.172 LaVerne, Boone & Mileski \|178 10.20401/rasi.3.2.172 24 knots to 20 knots generates an overall 42% reduction of fuel consumption, fuel cost, and CO2 emission” (Wong, Tai, Lau, & Raman, 2015). ( g ) The non-linear relationship between speed and fuel consumption looked at in regards to emissions also applies to operations costs and even the prospect of easing port congestion through slow steaming. “Costs and emissions may actually be even higher if a ship sails to a port at a maximum speed only to wait in line to enter once there” (Psaraftis & Kontovas, 2012). Burning less fuel, mitigating GHG, saving on fuel costs, minimizing engine maintenance, and easing port congestion can all be results of slow steaming as observed through various literature and studies. g While slow steaming has grown in popularity due to higher fuel prices and a higher presence of ecological awareness, it’s not the only solution associated with mitigation of carbon emissions. Studies and technologies are improving engines, implementing solar and wind power, and green routing and scheduling. Kontovas emphasizes that the research in the area of “Green Ship Routing and Scheduling” is just beginning to take off. Stating that with existing algorithms, such as the assignment algorithm, a reduction of emissions is possible (Kontovas, 2014). Mander in her work Slow steaming and a new dawn for wind propulsion: A multi-level analysis of two low carbon shipping transitions reviews the new technologies becoming available for other low carbon shipping methods such as wind propulsion. Stating that “Wind propulsion is at an earlier stage of innovation… and [can] drive the technology to the point that can challenge the existing regime” (Mander, 2016). With innovation around the corner and everyone joining on the slow steaming bandwagon, companies are looking for other ways to improve their emissions score card. Looking further into age, weight, and cargo carried could find small but effective efficiencies companies are searching for. The data was then placed by RightShip onto graphs in house and has been verified by other third parties mentioned above. Below we review the methodology of the calculations made by RightShip. Additionally, we compared the data received from RightShip by placing different parameters around the data. in which: • ME and AE, represent Main Engine(s) and Auxiliary Engine(s); • P, the power of the engines (kW); • CF, a conversion factor between fuel consumption and CO2 based on fuel carbon content; P, the power of the engines (kW); • CF, a conversion factor between fuel consumption and CO2 based on fuel carbon content; ; he certified specific fuel consumption of the engines (g/kWh); • SFC, the certified specific fuel consumption of the engines (g/kWh); • Capacity, the deadweight or gross tonnage (tonnes); • Vref, the ship speed (nm/h); and a correction factor to account for ship specific design elements (eg. ice-class). • fj, a correction factor to account for ship specific design elements (eg. ice-class). Ultimately, the equation reviews the emission produced (CO2) per nautical mile travelled. This is similar to how the calculation for EVDI below is found. This equation finds the emissions of an engine per distance traveled as well. This is different for the IMO’s EEDI, because it is only applicable new vessels. RASI, Volta Redonda/RJ, v. 3, n. 2, pp. 175-187, jul./dez. 2017. Data Collection Our data was collected by RightShip. They receive their data directly from the shipowners, managers, yards, classifications societies and charters. As of July 2017, RightShip collects data from eighty-three organizations, 52 of which represent 20% of global trade. Their data is evaluated by third parties to ensure authenticity, such as DNV GL. The sourced data is derived from Classification Societies (e.g. EEDI Technical Files), Ship sourced data (e.g. sea trial and shop tested supplied by the vessel owner/manager), Engine manufacturer’s specification (Sourced from ship yards), and IHS Maritime Database ( IMO publications). The data that is reviewed in this paper is specifically from Rightship’s Vetted fleet of 2015. These are the vessels that turned over their data and have been ensured to be authentic. The data from these ships were then placed into the EEDI or EVDI equations below to provide a rating for GHG emissions. The GHG emissions rating methodology stems from two sources: EEDI or EVDI. EEDI is the energy efficiency design index and EVDI is the existing vessel design index. EVDI is used when EEDI is added onto existing ships or new ones where EEDI is not available. EEDI is a regulatory requirement for new ships developed by IMO; EVDI was developed by RightShip. RightShip has provided the finished EVDI and GHG ratings. The EEDI equation below shows the emissions emitted by the engines and energy saving technology per transport work. RASI, Volta Redonda/RJ, v. 3, n. 2, pp. 175-187, jul./dez. 2017. http://www.rasi.uff.br http://www.rasi.uff.br LaVerne, Boone & Mileski \|179 10.20401/rasi.3.2.172 in which: Transport Work Energy Saving Technologies (Main Power) Energy Saving Technologies (Auxiliary Power) Main Engines Engines LaVerne, Boone & Mileski \|179 10.20401/rasi.3.2.172 Energy Saving Technologies (Main Power) Energy Saving Technologies (Auxiliary Power) Main Engines Main Engines Engines Transport Work Evaluation When comparing data, we modified the cargo carried and the age of the vessels. This allowed us to see the ages that have the highest GHG Emission ratings and the lowest EVDI. First, we looked at modifying only the cargo types. We were able to compare all dry cargoes, coal, iron ore and grain by the emissions rating and the EVDI. Next, we looked at modifying only age. This showed us what ages have the best EVDI and emissions ratings. The age ranges are 0-5, 6-10, 11-15, 16-20, and 21-355. The last 15 years years are not split into five year segments due to the small amount of data present within our parameters. EVDI and GHG Emissions Rating Scale RightShip collects data directly from the ships. They collect the distance traveled, engine capacity, fuel type, speed, carrying capacity and CO2 emissions. As shown in the equation above, to find EVDI, they took the CO2 emissions and divided that by the nautical miles traveled. This then gives a ratio of how much CO2 was produced per nautical mile. From there they are able to assign the GHG Emissions Rating by doing some calculations. First, they find the log values from the EVDI. Next, log values are weighted; then Z score is calculated from the weighted values. Lastly the scores are sized/ranked. These values are attributed to the total engine capacity, fuel type used, speed, and carrying capacity of each specific ship. EVDI are evaluated on a numerical scale where closer to 0 is a better score. The GHG Emissions Ratings are evaluated on an alphabetical scale; A being the best and G being the worst. Parameters selected Dry bulk carriers have many different characteristics to consider such as size in dead- weight tonnage (DWT), age in years, flag state (where a vessel flags out of), and cargo type. For our paper, we have looked at vessels with ages from 0-35 years old, DWT from 25,000 - 180,000 and cargo types are limited to grain, coal, and iron ore. We looked at the age range of 0-35 years old due to the average age of a dry bulk carrier is 25-27 years old (RightShip, 2015). The DWT was chosen due to the major vessel sizes operating in this sector are the Handy (<60,000), Panamax (60,000-89,000), and Cape size vessels (90,000+). We stopped our parameter of DWT at 180,000 because the average size of a Cape size bulker vessel is 156,000. Cargo types were limited to dry bulk with focus on grain, coal, and iron ore since the other sector was not defined. RASI, Volta Redonda/RJ, v. 3, n. 2, pp. 175-187, jul./dez. 2017. http://www.rasi.uff.br LaVerne, Boone & Mileski \|180 10.20401/rasi.3.2.172 LaVerne, Boone & Mileski \|180 10.20401/rasi.3.2.172 EVDI and GHG Emissions Rating Scale RASI, Volta Redonda/RJ, v. 3, n. 2, pp. 175-187, jul./dez. 2017. Results After defining our parameters we looked at the results. We found overall that 22% of ships within our parameters had an E,F, or G rating (Figure 1). http://www.rasi.uff.br http://www.rasi.uff.br http://www.rasi.uff.br LaVerne, Boone & Mileski \|181 10.20401/rasi.3.2.172 LaVerne, Boone & Mileski \|181 10.20401/rasi.3.2.172 Figure 1 Figure 1 When the vessels were categorized by their sizes, we found Handymax ships in the 60,000 DWT or less category to have the least amount of F and G rated ships, the most E rated ships, and the second most A rated ships. Panamax vessels from 60,000 DWT to 89,999 DWT had the most B and C rated vessels and the least amount of ships rated D through G. Finally the Capesize vessels, 90,000 DWT or greater, were found to have the most A rated vessels as well as the most F and G rated vessels. Overall, this shows that size of vessel does have a minor effect on the GHG emission ratings. When defining the cargo carried by ships, we found that all types of cargo (coal, iron ore, and grain) all had normally distributed curves. Some major findings were that the grain carrying ships had more A through D rated vessels at all sizes (Figure 2). RASI, Volta Redonda/RJ, v. 3, n. 2, pp. 175-187, jul./dez. 2017. http://www.rasi.uff.br Figure 2 Figure 2 RASI, Volta Redonda/RJ, v. 3, n. 2, pp. 175-187, jul./dez. 2017. http://www.rasi.uff.br http://www.rasi.uff.br RASI, Volta Redonda/RJ, v. 3, n. 2, pp. 175-187, jul./dez. 2017. LaVerne, Boone & Mileski \|182 10.20401/rasi.3.2.172 Iron ore carrying vessels had the most poorly rated vessels (E through G) at the Handy and Panamax sizes (Figure 3). Iron ore carrying vessels had the most poorly rated vessels (E through G) at the Handy and Panamax sizes (Figure 3). Figure 3 Coal carrying vessels had increasingly more E through G rated vessels as the size increased (Figure 4). Figure 3 Figure 3 Coal carrying vessels had increasingly more E through G rated vessels as the size increased (Figure 4). Figure 4 This data shows one is able to conclude that the cargo being carried in coordination with vessel size has an effect on the vessels GHG rating. As vessels increase in size the GHG emissions ratings accoiated with them improve. Figure 4 This data shows one is able to conclude that the cargo being carried in coordination with vessel size has an effect on the vessels GHG rating. As vessels increase in size the GHG emissions ratings accoiated with them improve. This data shows one is able to conclude that the cargo being carried in coordination with vessel size has an effect on the vessels GHG rating. As vessels increase in size the GHG emissions ratings accoiated with them improve. RASI, Volta Redonda/RJ, v. 3, n. 2, pp. 175-187, jul./dez. 2017. http://www.rasi.uff.br RASI, Volta Redonda/RJ, v. 3, n. 2, pp. 175-187, jul./dez. 2017. http://www.rasi.uff.br http://www.rasi.uff.br LaVerne, Boone & Mileski \|183 10.20401/rasi.3.2.172 Finally, we looked at the vessel age ranges. We found vessels 0-5 years old had 23% of the vessels rated E-G and 33% of vessels rated A-C (Figure 5). LaVerne, Boone & Mileski \|183 10.20401/rasi.3.2.172 Finally, we looked at the vessel age ranges. We found vessels 0-5 years old had 23% of the vessels rated E-G and 33% of vessels rated A-C (Figure 5). Finally, we looked at the vessel age ranges. We found vessels 0-5 years old had 23% of the vessels rated E-G and 33% of vessels rated A-C (Figure 5). Figure 5 Figure 5 Age range of 6-10 years old showed 23% of vessels rated E and F, none rated G and 29% rated A-C (Figure 6). Age range of 6-10 years old showed 23% of vessels rated E and F, none rated G and 29% rated A-C (Figure 6). RASI, Volta Redonda/RJ, v. 3, n. 2, pp. 175-187, jul./dez. 2017. http://www.rasi.uff.br Figure 6 Figure 6 Figure 6 RASI, Volta Redonda/RJ, v. 3, n. 2, pp. 175-187, jul./dez. 2017. http://www.rasi.uff.br RASI, Volta Redonda/RJ, v. 3, n. 2, pp. 175-187, jul./dez. 2017. http://www.rasi.uff RASI, Volta Redonda/RJ, v. 3, n. 2, pp. 175-187, jul./dez. 2017. RASI, Volta Redonda/RJ, v. 3, n. 2, pp. 175-187, jul./dez. 2017. http://www.rasi.uff.br LaVerne, Boone & Mileski \|184 10.20401/rasi.3.2.172 LaVerne, Boone & Mileski \|184 10.20401/rasi.3.2.172 Vessels 11-15 years old started to show a large gap in the sizes of vessels where no ships were recorded in the 100,000 DWT to 160,000 DWT range. The 11-15 year old vessels have no G rated vessels and 35% of vessels are A-C rated (Figure 7). Figure 7 16-20 years old vessels had 29% of A-C ratings, 26% of vessels rated E and F and no G rated vessels (Figure 8). Figure 7 16-20 years old vessels had 29% of A-C ratings, 26% of vessels rated E and F and no G rated vessels (Figure 8). RASI, Volta Redonda/RJ, v. 3, n. 2, pp. 175-187, jul./dez. 2017. http://www.rasi.uff.br Figure 8 Figure 8 Figure 8 Figure 8 RASI, Volta Redonda/RJ, v. 3, n. 2, pp. 175-187, jul./dez. 2017. http://www.rasi.uff. http://www.rasi.uff.br RASI, Volta Redonda/RJ, v. 3, n. 2, pp. 175-187, jul./dez. 2017. LaVerne, Boone & Mileski \|185 10.20401/rasi.3.2.172 LaVerne, Boone & Mileski \|185 10.20401/rasi.3.2.172 Starting at the age of 21 years old there are few ships represented in the age groups. For vessels 21-35 years old, there were 32% of ships rated A-C, and 25% are rated with F and G EVDI values (Figure 9). Figure 9 Overall the trend of larger ship sizes correlates with an improved EVDI is shown to be consistent with the cargo carried by the vessel data. Age also shows a strong role in emissions. As ships age the EVDI ratings overall decreased. Figure 9 Figure 9 Figure 9 Overall the trend of larger ship sizes correlates with an improved EVDI is shown to be consistent with the cargo carried by the vessel data. Age also shows a strong role in emissions. Analysis When analyzing the results, one can conclude that the DWT size class of the vessel does help define the GHG emissions rating. The next conclusion that can be made is that the cargo type does show tendencies that shipments of grain have better rated GHG Emissions. Finally, the age results show that there are larger than expected F and G rated vessels. Dry bulk carriers with age range from 21-35 had 16% of the vessels were G rated. The theory that quantity of E through G rated vessels increase as the age increases was proven. Lastly, the age range of 21-35 with the fewest number of vessels in the sample within the parameters showed only 32% of vessels were rated A through C. This suggests that these a large portion of ships run inefficiently. At this age, most ships are subject to more breakdowns and inefficient engines. These results also show that middle aged ships are the most efficient vessels. This suggests that they are producing less emissions per nautical mile. Figure 5 As ships age the EVDI ratings overall decreased. Overall the trend of larger ship sizes correlates with an improved EVDI is shown to be consistent with the cargo carried by the vessel data. Age also shows a strong role in emissions. As ships age the EVDI ratings overall decreased. 6. Conclusion and Remaining Questions Above you saw that the most efficient vessels are those carrying grain. Additionally the emissions ratings improved as vessel DWT increased. Vessels that from the ages 0-5 and http://www.rasi.uff.br http://www.rasi.uff.br RASI, Volta Redonda/RJ, v. 3, n. 2, pp. 175-187, jul./dez. 2017. LaVerne, Boone & Mileski \|186 10.20401/rasi.3.2.172 LaVerne, Boone & Mileski \|186 10.20401/rasi.3.2.172 16-20 had the best EVDI ratings compared to the other age classes. This was defined by the GHG emissions rating defined by EEDI or EVDI. These figures are found by measuring the CO2 emitted (work of the engine) per nautical mile. g p These leads us to look at the age composition of the fleet. Scraping ships at the age of 20 could improve overall fleet ratings. Discontinuing the use of poorly rated ships will improve emissions from the global fleet. Compositions of cargo carried should also be address. If vessels are able to find a mix of cargos that improves the emission ratings this would also improve the global fleet emissions. Weight was the surprising result. As DWT increased the overall emissions ratings increased. These larger vessels are believed to be the longer distance voyagers, thus improving the larger vessel emissions when compared to smaller vessels on shorter voyages. y g In our findings, we found overall that 22% of ships within our parameters had an E,F, or G rating. Seeing as RightShip’s data-collection covers approximately one-third of the world fleet, this is a large number of bulk carriers which need to improve their practices in order to assist in the goals that IMO would like to meet to reduce GHG emissions. Regulating agencies could also use data similar to this to see the need for more stringent regulations. From these graphs, we can see that even in the past 10 years, companies are still choosing to charter poorly rated (E, F & G) ships which inevitably costs them more money in fuel and external costs to the environment. We observed ships that were less than 10 years old, less than 5 years old, and even less than 3 years old still receiving these poor ratings. If no regulations are put into place and ships are still being built that have high environmental impacts, then we are not working toward reducing GHG emissions but instead just looking to make a quick buck. 6. Conclusion and Remaining Questions There could potentially be programs in place- “green practice incentive” programs- in order to reward those who are implementing practices such as slow steaming which are aiding in the reduction of GHG emissions. The ships with ratings A-D could find incentives through ports for their “green ship practices”. Not only would A-D rated ships benefit but hopefully this would also incentivize E-G rated ships to improve their practices so that they too can reap the benefits of green practice incentive programs. While we consider there are other options to how ships can get lower or higher emissions ratings such as route, weather, or time sensitive charters, the dry-bulk carriers are able to improve their GHG ratings by reducing speed and saving emissions and fuel. In future research, it would be helpful to be able to focus on dry-bulk carriers which have specific routes: transAtlantic and transPacific. Future research in this area could potentially lead to solving other issues, such as green ship routing. Acknowledgement The authors wish to thank Texas A&M University at Galveston and the Maritime Administration Department for the opportunity to research and develop this paper. The work in this paper would not have been possible without the data from RightShip. RightShip is an organization which has developed a systematic and transparent means of comparing the relative efficiency of the world’s shipping fleet. The Greenhouse Gas (GHG) Emissions Rating is an easy-to-use tool containing information on over 76,000 ocean going vessels. The GHG Rating is publically accessible via www.shippingefficiency.org. RASI, Volta Redonda/RJ, v. 3, n. 2, pp. 175-187, jul./dez. 2017. http://www.rasi.uff.br http://www.rasi.uff.br LaVerne, Boone & Mileski \|187 10.20401/rasi.3.2.172 RASI, Volta Redonda/RJ, v. 3, n. 2, pp. 175-187, jul./dez. 2017. References ALVAEZ, J.F., LONGVA, T., & ENGEBRETHSEN, E.S. (2010). A methodology to assess vessel berthing and speed optimization policies. Maritime Economics & Logistics 12(4): 327- 346. CARIOU, P. (2011). Is slow steaming a sustainable means of reducing CO2 emissions from container shipping? Transportation Research Part D: Transport and Environment, 16(3), 260-264. CHANG, C., & CHANG, C. (2013). Energy conservation for international dry bulk carriers via vessel speed reduction. Energy Policy, 59, 710-715. INTERNATIONAL MARITIME ORGANIZATION - IMO. (2014), Reduction of GHG Emissions from Ships, Third IMO GHG Study 2014 – Executive Summary Final Report, June 2014. KONTOVAS, C. (2014). The Green Ship Routing and Scheduling Problem: A conceptual approach. Transportation Research Part D 31, 61-69. PSARAFTIS, H. N., & KONTOVAS, C. A. (2012). Speed models for energy-efficient maritime transportation: A taxonomy and survey. Transportation Research Part C 26, 331- 351. PSARAFTIS, H. N., & KONTOVAS, C. A. (2014). Ship speed optimization: Concepts, models and combined speed-routing scenarios. Transportation Research Part C 44, 52-69. RIGHTSHIP. (2015). Sustainability Dashboard [graph]. Tableau. https://public.tableau.com/profile/rightship#!/vizhome/SustainabilityDashboard_0/VettedFleet -2015Dashboard WONG, E. Y., TAI, A. H., LAU, H. Y., & RAMAN, M. (2015). An utility-based decision support sustainability model in slow steaming maritime operations. Transportation Research Part E: Logistics and Transportation Review, 78, 57-69. RASI, Volta Redonda/RJ, v. 3, n. 2, pp. 175-187, jul./dez. 2017. http://www.rasi.uff.br
https://openalex.org/W2797342218	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0195636&type=printable	English	null	Protein subnuclear localization based on a new effective representation and intelligent kernel linear discriminant analysis by dichotomous greedy genetic algorithm	PloS one	2,018	cc-by	10,707	RESEARCH ARTICLE Protein subnuclear localization based on a new effective representation and intelligent kernel linear discriminant analysis by dichotomous greedy genetic algorithm Shunfang Wang☯, Yaoting Yue☯ School of Information Science and Engineering, Yunnan University, Kunming, PR China a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 ☯These authors contributed equally to this work. sfwang_66@ynu.edu.cn Abstract A wide variety of methods have been proposed in protein subnuclear localization to improve the prediction accuracy. However, one important trend of these means is to treat fusion representation by fusing multiple feature representations, of which, the fusion process takes a lot of time. In view of this, this paper novelly proposed a method by combining a new single feature representation and a new algorithm to obtain good recognition rate. Specifically, based on the position-specific scoring matrix (PSSM), we proposed a new expression, cor- relation position-specific scoring matrix (CoPSSM) as the protein feature representation. Based on the classic nonlinear dimension reduction algorithm, kernel linear discriminant analysis (KLDA), we added a new discriminant criterion and proposed a dichotomous greedy genetic algorithm (DGGA) to intelligently select its kernel bandwidth parameter. Two public datasets with Jackknife test and KNN classifier were used for the numerical experi- ments. The results showed that the overall success rate (OSR) with single representation CoPSSM is larger than that with many relevant representations. The OSR of the proposed method can reach as high as 87.444% and 90.3361% for these two datasets, respectively, outperforming many current methods. To show the generalization of the proposed algo- rithm, two extra standard datasets of protein subcellular were chosen to conduct the expending experiment, and the prediction accuracy by Jackknife test and Independent test is still considerable. Editor: Xiangtao Li, Northeast Normal University, CHINA Received: December 15, 2017 Accepted: March 26, 2018 Published: April 12, 2018 Copyright: © 2018 Wang, Yue. 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 came from previous studies, whose links are gathered in the GitHub platform at the following URL: https://github.com/tingyaoyue/Dataset.git". OPEN ACCESS Citation: Wang S, Yue Y (2018) Protein subnuclear localization based on a new effective representation and intelligent kernel linear discriminant analysis by dichotomous greedy genetic algorithm. PLoS ONE 13(4): e0195636. https://doi.org/10.1371/journal. pone.0195636 Editor: Xiangtao Li, Northeast Normal University, CHINA Subnuclear localization with new representation and kernel linear discriminant analysis dramatically day by day so that those traditional experimental methods can’t satisfy the demands of current researches on account of their low efficiency and highly cost. Therefore, in order to manage and address these huge biological data, computational techniques are essen- tial. There are two typical procedures when researchers apply machine learning methods to predict protein subnuclear location. One is to construct good representations for collecting as much protein sequence information as possible and the other is to develop effective models for prediction and classification [3, 4]. Competing interests: The authors have declared that no competing interests exist. As far as feature representations are concerned, Nakashima and Nishikawa proposed a well-known representation, amino acid composition (AAC) [5], which describes the occurrence frequency of 20 kinds of essential amino acids in a protein sequence. However, AAC ignores the associated information among amino acids [4]. Therefore, dipeptide composition (DipC) was presented by considering 400 components of dipeptide composition information along local order of amino acids [6]. Nevertheless, the discrimination of DipC is still insufficient. Sub- sequently, taking into account both amino acid composition information and amphipathic sequence-order information, Chou et al. introduced the pseudo-amino acid composition (PseAAC), and relevant experimental results proved that the discriminant performance of PseAAC overmatched both AAC and DipC partly [7–11]. Afterwards, the position-specific scoring matrix (PSSM) was proposed by considering the evolution information of amino acids, and PSSM is more helpful than PseAAC for protein subnuclear localization [12]. But the predic- tion accuracy still can’t meet researchers’ expectation. Hence, they tried to build more efficient protein feature expression. Based on above single feature representations, researchers proposed the concept of fusion representation by combining two single expressions for improving the prediction accuracy since fusion representation contained more original protein sequence information [4, 13, 14]. However, although the predictive accuracy is improved with this kind of method, the extra workload and time-consuming caused by the process of fusing different representations increase a lot [4]. With this consideration, the research of this paper devoted to developing a new single representation to make it can express protein sequence more effectively, and then lots of time will be saved by doing so relative to the fusion representation. Introduction Funding: This research is supported by grants from National Natural Science Foundation of China (11661081) and Natural Science Foundation of Yunnan Province (2017FA032). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Subnuclear localization of protein is very important for molecular cell biology, proteomics, and drug discovery and so on [1, 2]. When the basic function of a protein is known, the infor- mation about its location in the cell nucleus may indicate some important facts such as the pathway an enzyme belongs to [2]. Thus, if proteins are located at wrong positions in the nucleus or in a cell, some diseases, even cancer, will be caused. With the development of human genome project and proteomics project, numerous protein sequences increase PLOS ONE \| https://doi.org/10.1371/journal.pone.0195636 April 12, 2018 1 / 20 PLOS ONE \| https://doi.org/10.1371/journal.pone.0195636 April 12, 2018 Subnuclear localization with new representation and kernel linear discriminant analysis almost doesn’t have to train, which will save a certain amount of time. In practical application, the calculated quantity of KNN classifier is proportional to the sample size [32]. Here in this paper, the experimental data are small sample size, which makes it more appropriate to utilize the KNN classifier compared to those classifiers mentioned above. Hence, we only employ the simple KNN classifier in this paper to both reduce the computational complexity and highlight the innovation of the proposed method, CoPSSM with intelligent KLDA based on DGGA. To sum up, although good results were obtained based on those above approaches, namely, fusing different representations, developing more effective models or classifiers, shortcomings still exist in current works. Computation complexity, for instance, increases a lot to some extent. So, if we can improve the prediction accuracy of protein subnuclear location only use the single feature representation and simple classifier, a lot of time-consuming and costly work will be saved, and that will be very meaningful. And here, the work of this paper is just to realize this goal. First of all steps, the single feature vector, position-specific scoring matrix (PSSM), was extracted from the given original protein sequence and then a new feature ex- pression CoPSSM would be created based on the PSSM matrix. Next, the nonlinear dimen- sionality reduction (DR) algorithm, kernel linear discriminant analysis (KLDA), whose bandwidth parameter was intelligently optimized by the proposed new discriminant criterion and dichotomous greedy genetic algorithm (DGGA), has been employed to address the high- dimensionality problem by transforming the representation of protein sequence for arriving at an optimal expression for K-nearest-neighbor (KNN) classifier. The final numerical experi- mental results with Jackknife test show our proposed single feature representation CoPSSM and optimization algorithm are efficient in the prediction of protein subnuclear location. p g p p Here, we listed abbreviation of the full name for all terms appeared in this paper in Table 1. https://doi.org/10.1371/journal.pone.0195636.t001 PLOS ONE \| https://doi.org/10.1371/journal.pone.0195636 April 12, 2018 Next, due to the high dimensionality of protein feature representation data, lots of dimen- sion reduction algorithms were employed to extract feature such as linear discriminate analysis (LDA) [4, 13, 15], principal component analysis (PCA), kernel principal component analysis (KPCA) [12], kernel entropy component analysis (KECA), kernel LDA (KLDA) and so on [12, 16–22]. Since the nonlinear characteristics are more popular than the linear characteristics in biology [12, 23, 24], the nonlinear kernel algorithms are the keystone of this paper. Especially, KLDA is selected to use because it not only can reduce dimensionality but also can help classi- fication and recognition. However, the window width parameter of kernel function in current studies tends to be empirically selected, which is not reasonable. So instead, in this paper, we realized the intelligent selection of the bandwidth parameter by our proposed new discrimi- nant criterion and optimization algorithm DGGA. After extracting good features, researchers try to develop effective classifiers for prediction, including biological neural networks, Bayes- ian networks, support vector machines (SVM), ensemble-classifiers [25, 26], optimally weighted fuzzy K-NN algorithm [2] and so on. Thereinto, the neural network not only needs mass of data to train the structural classifier model, but also needs effective method and plenty of time to tune the network parameters, which is a hard problem to be improved [27]. Simi- larly, the prediction results based on Bayes discriminant algorithm can be improved as the amount of training data increases; therefore, a small number of training data may cause the prediction results less stable [28]. In addition, although the SVM, ensemble-classifier and the weighted fuzzy K-NN algorithm can get good prediction, they are all time-consuming in the training phase [25, 29–31]. Compared with them, the KNN classifier is lazy learning, namely, it PLOS ONE \| https://doi.org/10.1371/journal.pone.0195636 April 12, 2018 2 / 20 Dataset To validate the adaptability and the efficiency of the proposed method in this paper, and to have a critical comparison with other studies, two public benchmark datasets were chosen to Table 1. Abbreviation for the corresponding term. Table 1. Abbreviation for the corresponding term. Number Full Name of Term Abbreviation 1 Pseudo-amino acid composition PseAAC 2 Position-specific scoring matrix PSSM 3 Correlation position-specific scoring matrix CoPSSM 4 Kernel linear discriminant analysis KLDA 5 K-nearest-neighbor KNN 6 Genetic algorithm GA 7 Dichotomous greedy genetic algorithm DGGA 8 Overall success rate OSR 9 True positive TP 10 True negative TN 11 False positive FP 12 False negative FN 13 Sensitivity SE 14 Specificity SP 15 Accuracy ACC 16 Mathew’s correlation coefficient MCC 3 / 20 PLOS ONE \| https://doi.org/10.1371/journal.pone.0195636 April 12, 2018 PLOS ONE \| https://doi.org/10.1371/journal.pone.0195636 April 12, 2018 Subnuclear localization with new representation and kernel linear discriminant analysis Table 2. Constitutions of protein benchmark datasets. Dataset 1 of ten subnuclear locations Dataset 2 of nine subnuclear locations Class Subnuclear Location Number Class Subnuclear Location Number 1 Centromere 86 1 Chromatin 99 2 Chromosome 113 2 Heterochromatin 22 3 Nuclear envelope 17 3 Nuclear envelope 61 4 Nuclear matrix 18 4 Nuclear matrix 29 5 Nuclear pore complex 12 5 Nuclear pore complex 79 6 Nuclear speckle 50 6 Nuclear speckle 67 7 Nucleolus 294 7 Nucleolus 307 8 Nucleoplasm 30 8 Nucleoplasm 37 9 Telomere 37 9 Nuclear PML body 13 10 Nuclear PML body 12 Sum 669 Sum 714 https://doi.org/10.1371/journal.pone.0195636.t002 Table 2. Constitutions of protein benchmark datasets. conduct the numerical experiments. The first dataset was constructed by Ravindra Kumar et al. in 2014 [33], which is in their web-server named SubNucPred for predicting protein sub- nuclear localization with the link of http://proteininformatics.org/mkumar/subnucpred/index. html. The second dataset is in web-sever Nuc-Ploc [7], which was constructed by Shen and Chou in 2007 and could be downloaded from the link of http://www.csbio.sjtu.edu.cn/bioinf/ Nuc-PLoc/Supp-A.pdf. Detailed information of these two datasets is in Table 2. As shown in Table 2, dataset 1 totally contains 669 proteins that attribute to 10 subnuclear localizations and dataset 2 contains 714 proteins in total and locates at 9 subnuclear localizations. localizations and dataset 2 contains 714 proteins in total and locates at 9 subnuclear localizations. Next, to show the generalization of the proposed method, two protein subcellular bench- mark datasets were chosen to conduct the expending experiment, as are shown in Table 3. Next, to show the generalization of the proposed method, two protein subcellular bench- mark datasets were chosen to conduct the expending experiment, as are shown in Table 3. From Table 3, it can be found that datasets 3 and 4 contain 653 and 643 proteins, respec- tively, and they can be downloaded directly from http://www.csbio.sjtu.edu.cn/bioinf/Gneg/ Data.htm [34]. To sum up, in this paper, we used four datatsets that were constructed in previous studies [7, 33, 34]. For an easy access to all these data, we construct a new link to gather all the link information about these datasets, that is https://github.com/tingyaoyue/Dataset.git. https://doi.org/10.1371/journal.pone.0195636.t002 A newly proposed feature representation CoPSSM Hence, the position-specific scor- ing matrix (PSSM) was introduced to represent the evolution information of a protein sam- ple P with L amino acid residues. Its descriptor is shown as following: PPSSM ¼ M1!1 M1!2 M1!20 M2!1 M2!2 M2!20 ... ... .. . ... ML!1 ML!2 ML!20 2 6666664 3 7777775 ð1Þ ð1Þ where Mi!j(i = 1,2,. . .,L; j = 1,2,. . .,20) represents the score of the amino acid residue in the i th position of the protein sequence being replaced by the amino acid type j during the evo- lution process. And here in this paper, the PPSSM matrix was generated via using PSI-BLAST to search the Swiss-Prot database, of which the iterative times were 3 and the E-value was 0.001. where Mi!j(i = 1,2,. . .,L; j = 1,2,. . .,20) represents the score of the amino acid residue in the i th position of the protein sequence being replaced by the amino acid type j during the evo- lution process. And here in this paper, the PPSSM matrix was generated via using PSI-BLAST to search the Swiss-Prot database, of which the iterative times were 3 and the E-value was 0.001. 3. Introduction for the newly proposed feature representation CoPSSM A newly proposed feature representation CoPSSM Before introducing the proposed new feature expression CoPSSM, we first need to give some brief presentation for PseAAC and PSSM that are used for comparison in this paper. Then, introduction for CoPSSM will be deployed based on PSSM. Table 3. Constitutions of protein benchmark datasets for expending experiment. Class Subcellular Location Number of Dataset 3 Number of Dataset 4 1 Cytoplasm 152 210 2 Extracell 76 20 3 Fimbrium 12 4 4 Flagellum 6 1 5 Inner membrane 186 345 6 Nucleoid 6 1 7 Outer membrane 103 13 8 Periplasm 112 49 Sum 653 643 https://doi.org/10.1371/journal.pone.0195636.t003 Table 3. Constitutions of protein benchmark datasets for expending experiment. 4 / 20 PLOS ONE \| https://doi.org/10.1371/journal.pone.0195636 April 12, 2018 PLOS ONE \| https://doi.org/10.1371/journal.pone.0195636 April 12, 2018 Subnuclear localization with new representation and kernel linear discriminant analysis 1. PseAAC, put forward by Chou et al., represents a protein sequence with its sequence com- position and order information in a vector [7]. Generally speaking, PseAAC is expressed as PPseAAC = [p1,p2,. . .,p20,p20+1,. . .,p20+2β]T. And here, the parameter β is set as 10 empirically to obtain a 40-D feature vector. The first 20 components reflect the effect of the classical 20 amino acid composition, and components from 20 + 1 to 20 + 2β reflect the amphipathic sequence-order pattern with considering the impact of hydrophobic and hydrophilic of amino acids [35–38]. 2. PSSM, whose description is as below: A variety of variations, such as the insertion, substitution or deletion of one or several amino acid residues in the protein sequence, often occur in the biological evolution process. And with long-term accumulation of these variations, similarities between the original and the new synthesis proteins are reducing gradually, but these homologous proteins may exhibit remarkably similar structures and functions [39]. Hence, the position-specific scor- ing matrix (PSSM) was introduced to represent the evolution information of a protein sam- ple P with L amino acid residues. Its descriptor is shown as following: p A variety of variations, such as the insertion, substitution or deletion of one or several amino acid residues in the protein sequence, often occur in the biological evolution process. And with long-term accumulation of these variations, similarities between the original and the new synthesis proteins are reducing gradually, but these homologous proteins may exhibit remarkably similar structures and functions [39]. PLOS ONE \| https://doi.org/10.1371/journal.pone.0195636 April 12, 2018 3. Introduction for the newly proposed feature representation CoPSSM Feature representation plays an important role in protein subnuclear localization [4]. Based on this idea, this paper skillfully proposed a new feature expression. Since sizes of above obtained PPSSM matrices were not unified for different proteins, researchers usually trans- formed them into 400-D vectors by adding all rows of the same element [12, 13]. Here, we will develop a better representation. Firstly, calculate average value of each column in PPSSM according to (2). M j ¼ 1 L PL i¼1Mi!j ðj ¼ 1; 2; . . . 20Þ ð2Þ ð2Þ Secondly, calculate the product of two different elements in the above obtained 20 average values according to formula (3). Secondly, calculate the product of two different elements in the above obtained 20 average values according to formula (3). CoPSSM ¼ Mj Mk ðj ¼ 1; 2; . . . ; 20; k is integer and j k 20Þ ð3Þ ð3Þ Last, a 210-D vector shown in (4) will be attained according to Eqs (2) and (3). Since it takes the correlation of two different average values into consideration, we named the pro- posed feature representation as correlation position-specific scoring matrix CoPSSM. CoPSSM ¼ ðCoPSSM1; CoPSSM2; . . . ; CoPSSM210Þ ð4Þ ð4Þ 5 / 20 PLOS ONE \| https://doi.org/10.1371/journal.pone.0195636 April 12, 2018 Subnuclear localization with new representation and kernel linear discriminant analysis A newly proposed discriminant criterion added to KLDA Hence, we defined the above ratio and stipulated that the best reduced data corresponded to the largest ratio. Hence, formula (5) is set as the fitness function of the proposed dichotomous greedy genetic algorithm (DGGA). Thereinto, DW and DB can be obtained via formulas (6) and (7). respectively. Our purpose here is to minimize DW and to maximize DB. Nevertheless, they two will change randomly during the course of the experiment. Hence, we defined the above ratio and stipulated that the best reduced data corresponded to the largest ratio. Hence, formula (5) is set as the fitness function of the proposed dichotomous greedy genetic algorithm (DGGA). Thereinto, DW and DB can be obtained via formulas (6) and (7). DW ¼ PK i¼1 PNi m¼1kri m aik2 ð6Þ DB ¼ PK i¼1kai ak2 ð7Þ DW ¼ PK i¼1 PNi m¼1kri m aik2 ð6Þ K DW ¼ PK i¼1 PNi m¼1kri m aik2 ð6Þ ð6Þ DB ¼ PK i¼1kai ak2 ð7Þ DB ¼ PK i¼1kai ak2 ð7Þ ð7Þ where the 2-norm kk2 denotes the Euclidean distance, K is the number of protein type, Ni is the number of class i, ai represents the mean vector of class i and a denotes the mean vector of all classes, i.e., ai ¼ 1 Ni PNi m¼1 ri m and a ¼ 1 N PN n¼1 rn. where the 2-norm kk2 denotes the Euclidean distance, K is the number of protein type, Ni is the number of class i, ai represents the mean vector of class i and a denotes the mean vector of all classes, i.e., ai ¼ 1 Ni PNi m¼1 ri m and a ¼ 1 N PN n¼1 rn. A newly proposed discriminant criterion added to KLDA Kernel linear discriminant analysis, also known as generalized discriminant analysis is the ker- nel extension method of linear discriminant analysis (LDA), which is expanded to solve the nonlinear problems. Therefore, KLDA is much suitable for processing biological data because of its high-dimensional and nonlinear characteristics. KLDA algorithm maps the input vectors to a higher dimensional feature space F via the nonlinear mapping function ;, and then it exe- cutes the linear discriminant analysis in the high dimensional feature space [40]. To increase further understanding, the KLDA algorithm will be described in detail in the S1 File, in which, these two literatures [41, 42] will be cited. But what actually matters is the bandwidth parameter of the kernel function, which changes the mapping relation between the input space and the feature space so that it can affect the properties of the feature space. So far, there is a lack of good methods to find out the best value of the window width parameter [40, 43]. Usually, researchers set this parameter empirically. Besides, a method called grid searching method was used to determine value of this parameter [44]. But they were partly irrational for lacking of a rational answer even good results were obtained, and there still existed defects in the grid searching method, missing the valid values and much time-consuming for instance. Thus, we try to introduce more reasonable method to select the bandwidth parameter to deal with this problem in protein subnuclear location. Here in this paper, the gauss kernel function: K x; y ð Þ ¼ exp kxyk2 2s2 is taken into consideration, and we’ll propose a new discriminant criterion to evaluate its bandwidth parameter σ whether good or not. and we’ll propose a new discriminant criterion to evaluate its bandwidth parameter σ whether good or not. Providing that the reduced samples by KLDA are r1,r2,. . .,rN, then we define a new discrim- inant criterion to evaluate distinguishability of these reduced data, as formula (5): max DB DW ð5Þ ð5Þ where DW and DB represents the within-class dispersion and the between-class dispersion, where DW and DB represents the within-class dispersion and the between-class dispersion, respectively. Our purpose here is to minimize DW and to maximize DB. Nevertheless, they two will change randomly during the course of the experiment. An improved dichotomous greedy genetic algorithm (DGGA) for kernel parameter selecting Genetic algorithm (GA) is a kind of adaptive method for dealing with complex optimization problem whose core is the “survival of the fittest” rule and chromosomal crossover mechanism within a group. Increasingly wide attention and applications are drawn in GA for its robust- ness, parallelism and global optimization characteristics in recent years. Whereas 6 / 20 PLOS ONE \| https://doi.org/10.1371/journal.pone.0195636 April 12, 2018 Subnuclear localization with new representation and kernel linear discriminant analysis shortcomings still exist in this algorithm such as easy to trap in local optimum. Therefore, we proposed a new algorithm, dichotomous greedy genetic algorithm (DGGA), to improve gen- eral GA algorithm. DGGA is based on GA by introducing the idea of inter-partition and Greedy Algorithm. In simple terms, in order to search the gauss kernel parameter more effi- ciently, we keep on dividing the interval into two subintervals and reserving the effective one on which the largest fitness is obtained by employing GA, which derives from the theory of Greedy Algorithm until the iterations run out. Thus, the proposed DGGA is named as dichot- omous greedy genetic algorithm, of which, the word dichotomous means dividing the interval into two subintervals continually and greedy signifys the using of the idea of Greedy Algo- rithm. The specific steps of DGGA are listed as below. • Step1: Select a certain amount of points randomly in the given interval [X0,Xn], as the initial population; • Step2: Calculate fitness of the initial population; • Step2: Calculate fitness of the initial population; • Step3: Let Xmax be the location identifier of the point with maximal fitness among the initial population, then we get 2 inter-partitions [X0,Xmax] and [Xmax,Xn]; • Step4: Generate the initial population P1 and P2 randomly in the inter-partition of [X0,Xmax] and [Xmax,Xn] respectively, then calculate their fitness respectively, named f1 and f2; • Step5: Employed GA to optimize the kernel parameter with (f1,P1) and (f2,P2) as the input parameters respectively, then the updated population and fitness are marked as (newf1, newP1) and (newf2, newP2); • Step6: Let maxfit1 be the max value of newf1 and maxfit2 be the max value of newf2. • Step7: If maxfit1 is larger than maxfit2, let X1 be the corresponding location identifier and make Xn = Xmax, then let Xmax = X1; else, let X2 be the corresponding location identifier of maxfit2 and make X0 = Xmax, then let Xmax = X2. An improved dichotomous greedy genetic algorithm (DGGA) for kernel parameter selecting Therefore, the updated inter-partitions are as [X0,Xmax] and [Xmax,Xn]. Next, turn to Step4 until the iterations run out. At last, to have an intuitivism apprehension of DGGA, we summarize the above procedures and give the full design flow of DGGA for optimizing the kernel parameter, displayed in Fig 1. After the optimal kernel parameter had been trained, it would be realizable to calculate the optimal projection matrix, and then both train dataset and test dataset would be mapped to the low-dimensional feature space. Last, the KNN classifier would be employed to predict cor- responding protein subnuclear location according to the rule of Jackknife test. We’ll provide Fig 2 to display specific flows of the whole processes predicting protein subnuclear location. PLOS ONE \| https://doi.org/10.1371/journal.pone.0195636 April 12, 2018 PLOS ONE \| https://doi.org/10.1371/journal.pone.0195636 April 12, 2018 Assessment criteria, classifier and test method PLOS ONE \| https://doi.org/10.1371/journal.pone.0195636 April 12, 2018 8 / 20 Subnuclear localization with new representation and kernel linear discriminant analysis Fig 2. The flowchart for predicting protein subnuclear location. Fig 2. The flowchart for predicting protein subnuclear location. https://doi.org/10.1371/journal.pone.0195636.g002 https://doi.org/10.1371/journal.pone.0195636.g002 https://doi.org/10.1371/journal.pone.0195636.g002 In addition, we also defined the overall success rate (OSR) as follow to evaluate the overall classification effects. From Eqs (8) to (12), k is the number of protein type. OSR ¼ Xk i¼1TPðiÞ Xk i¼1ðTPðiÞ þ FNðiÞÞ ð12Þ ð12Þ Last, in this paper, we take KNN as the classifier for its simplicity, but competitive results. The Cosine distance is used to measure the close degree of two proteins. Besides, Jackknife test which is accounted as the most reasonable testing method are employed to estimate the predic- tion performance of our proposed method. Assessment criteria, classifier and test method To evaluate prediction performance of the proposed method, indexes: Sensitivity (SE), Speci- ficity (SP), ACC (Accuracy) and MCC (Mathew’s Correlation Coefficient) are calculated to compare different representations in the case of Jackknife test. In the following formulas (8)– (12), TP means the true positive and TN means the true negative, of which both are the num- ber of proteins that were correctly located, while FP (the false positive) and FN (the false nega- tive) are the number of those that were wrongly located proteins[4]. Then, 4 index equations are obtained: SEðiÞ ¼ TPðiÞ=ðTPðiÞ þ FNðiÞÞ ði ¼ 1; 2; . . . ; kÞ ð8Þ SPðiÞ ¼ TNðiÞ=ðTNðiÞ þ FPðiÞÞ ði ¼ 1; 2; . . . ; kÞ ð9Þ SEðiÞ ¼ TPðiÞ=ðTPðiÞ þ FNðiÞÞ ði ¼ 1; 2; . . . ; kÞ ð8Þ ð8Þ ð9Þ 7 / 20 PLOS ONE \| https://doi.org/10.1371/journal.pone.0195636 April 12, 2018 Subnuclear localization with new representation and kernel linear discriminant analysis Fig 1. The DGGA algorithm for searching kernel parameter. https://doi.org/10.1371/journal.pone.0195636.g001 Fig 1. The DGGA algorithm for searching kernel parameter. Fig 1. The DGGA algorithm for searching kernel parameter. https://doi.org/10.1371/journal.pone.0195636.g001 ACCðiÞ ¼ TPðiÞ þ TNðiÞ TPðiÞ þ FPðiÞ þ TNðiÞ þ FNðiÞ ði ¼ 1; 2; . . . ; kÞ ð10Þ MCCðiÞ ¼ ðTPðiÞ TNðiÞÞ ðFPðiÞ FNðiÞÞ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ ðTPðiÞ þ FPðiÞÞ ðTPðiÞ þ FNðiÞÞ ðTNðiÞ þ FPðiÞÞ ðTNðiÞ þ FNðiÞÞ p ði ¼ 1; 2; . . . ; kÞð11Þ ACCðiÞ ¼ TPðiÞ þ TNðiÞ TPðiÞ þ FPðiÞ þ TNðiÞ þ FNðiÞ ði ¼ 1; 2; . . . ; kÞ ð10Þ ð10Þ MCCðiÞ ¼ ðTPðiÞ TNðiÞÞ ðFPðiÞ FNðiÞÞ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ ðTPðiÞ þ FPðiÞÞ ðTPðiÞ þ FNðiÞÞ ðTNðiÞ þ FPðiÞÞ ðTNðiÞ þ FNðiÞÞ p ði ¼ 1; 2; . . . ; kÞð11Þ Here, SE, also called the success rate, denotes the rate of positive samples correctly located; SP denotes the rate of negative samples correctly located and ACC means the rate of correctly located samples. MCC returns a value lying in [–1, 1] and the value of MCC reflects the predic- tion consequences. The value of 1 denotes a perfect prediction, 0 represents random prediction and -1 represents a bad prediction. Generally, MCC is regarded as one of the best assessment indexes [45]. Comparison results of the newly proposed single expression CoPSSM with two other common representations PseAAC and PSSM To demonstrate effectiveness of the newly proposed single feature expression CoPSSM, we conducted the comparison experimental investigation. Firstly, the often-used 40-D PseAAC and 400-D PSSM were extracted from the given protein sequences respectively. Secondly, the proposed 210-D CoPSSM would be obtained based on PSSM matrix. Thirdly, the KNN classi- fier with cosine distance was used to predict protein subnuclear location. Last, the Jackknife test, identified as the most objective and rigorous method was utilized to evaluate the classifica- tion performance [14]. Concrete prediction accuracy (ACC) for each Class and the overall suc- cess rate (OSR) are in Tables 4 and 5. From Table 4, it clearly displays that the overall prediction success rate for our newly pro- posed feature representation CoPSSM outperforms the two most-frequently used expressions PseAAC and PSSM. Not only that, but CoPSSM can resolve the imbalanced data problem. As evident in Table 2, these two benchmark datasets are heavily imbalanced. Generally, classifier tends to be biased towards the majority class, resulting in poor accuracy for those classes hav- ing smaller number of samples [33]. For PseAAC, we can find bad prediction for Classes 8 and 10 with accuracy of 0, and this kind of situation still exists in PSSM although its OSR is larger than that of PseAAC. For CoPSSM, even though some prediction accuracies are smaller than those of PseAAC and PSSM for the same Class, there is no bad prediction, which denotes the proposed new feature representation CoPSSM can solve the data imbalance problem to a cer- tain degree. This is because CoPSSM can express protein sequence better than the 40-D PseAAC and the 400-D PSSM. Table 5 also shows that the proposed CoPSSM performs better than both PseAAC and PSSM. Besides, we can clearly see that bad prediction accuracies for Classes 8 and 9 are all equal to 0 when the feature representation is PseAAC. For CoPSSM, even though several pre- diction accuracies are smaller than those of PseAAC and PSSM, there is no bad prediction, Table 4. Prediction results for protein subnuclear of dataset 1. Results and discussion In this paper, the number of the initial population and the iterations were selected as 10 with taking the calculating time into consideration. Larger population and bigger iterations will cost more time undoubtedly, while smaller population and iterations may cause incomplete PLOS ONE \| https://doi.org/10.1371/journal.pone.0195636 April 12, 2018 9 / 20 Subnuclear localization with new representation and kernel linear discriminant analysis optimization. Next, we empirically set probability of the selection, crossover and mutation operator as 0.5, 0.7 and 0.1 respectively. Finally, the hardware operating environment of this paper is: Intel(R) Core(TM) i7-3770 CPU @3.40GHz 3.40 GHz, RAM 4G, Matlab R2011b. Class 1 ~ Class 10 denote Centromere, Chromosome, Nuclear envelope, Nuclear matrix, Nuclear pore complex, Class 1 ~ Class 10 denote Centromere, Chromosome, Nuclear envelope, Nuclear matrix, Nuclear pore complex, Nuclear speckle Nucleolus Nucleoplasm Telomere and Nuclear PML body respectively Nuclear speckle, Nucleolus, Nucleoplasm, Telomere and Nuclear PML body respectively. https://doi.org/10.1371/journal.pone.0195636.t004 https://doi.org/10.1371/journal.pone.0195636.t004 PLOS ONE \| https://doi.org/10.1371/journal.pone.0195636 April 12, 2018 Class 1 ~ Class 10 denote Centromere, Chromosome, Nuclear envelope, Nuclear matrix, Nuclear pore complex, Nuclear speckle, Nucleolus, Nucleoplasm, Telomere and Nuclear PML body respectively. Results of assessment criteria for PseAAC, PSSM and the proposed CoPSSM Next, to objectively evaluate effectiveness of our proposed new feature representation CoPSSM in protein subnuclear location, we calculated the comparative values of these 4 assessment cri- teria: SE (Sensitivity), SP (Specificity), ACC (Accuracy) and MCC (Mathew’s Correlation Coefficient) for PseAAC, PSSM and the proposed CoPSSM. What shown in Table 6 is compar- ison result for dataset 1, and Table 7 is results for dataset 2. Tables 6 and 7 were obtained according to Eqs (8)–(11). In Table 6, we can learn that there exist such outliers of 0 and “-” for the expressions PseAAC and PSSM, where “-” means the missing phenomenon in computation. According to Eqs (8) and (11), it can be inferred that none of these types of proteins were correctly located and no other type of proteins was misclassified to them, which caused the exceptional values 0 and “-”, respectively. Indeed, the results in Table 4 confirm this conclusion. In Table 6, we can see most values of CoPSSM are larger than those of PseAAC and PSSM for the same Class, which signifys that CoPSSM outperforms them. For the outliers 0 and “-” in Table 7, the explanation is the same as that of Table 6, and the results in Table 5 can confirm it. Similarly, we can conclude that the proposed feature repre- sentation CoPSSM performs better than both PseAAC and PSSM in protein subnuclear localization. Comparison results of the newly proposed single expression CoPSSM with two other common representations PseAAC and PSSM Type Feature Representation PseAAC (40-D) PSSM (400-D) The proposed CoPSSM (210-D) Class 1 30.2326% 55.8140% 70.9302% Class 2 36.2832% 41.5929% 53.0973% Class 3 29.4118% 0 23.5294% Class 4 16.6667% 22.2222% 50% Class 5 41.6667% 58.3333% 66.6667% Class 6 32% 30% 26% Class 7 86.0544% 81.6327% 78.5714% Class 8 0 10% 23.3333% Class 9 5.4054% 10.8108% 43.2432% Class 10 0 0 25% OSR 52.4663% (K = 9) 55.0075% (K = 5) 61.5845% (K = 1) Table 4. Prediction results for protein subnuclear of dataset 1. Table 4. Prediction results for protein subnuclear of dataset 1. 10 / 20 PLOS ONE \| https://doi.org/10.1371/journal.pone.0195636 April 12, 2018 PLOS ONE \| https://doi.org/10.1371/journal.pone.0195636 April 12, 2018 10 Subnuclear localization with new representation and kernel linear discriminant analysis Table 5. Prediction results for protein subnuclear of dataset 2. Type Feature Representation PseAAC (40-D) PSSM (400-D) The proposed CoPSSM (210-D) Class 1 48.4848% 69.6970% 59.5960% Class 2 22.7272% 36.3636% 36.3636% Class 3 24.5902% 29.5082% 49.1803% Class 4 13.7931% 31.0345% 44.8276% Class 5 55.6962% 67.0886% 78.4810% Class 6 29.8507% 40.2985% 32.8358% Class 7 81.4332% 71.3355% 79.1531% Class 8 0 16.2162% 18.9189% Class 9 0 7.6923% 15.3846% OSR 54.0616% (K = 12) 57.4230% (K = 3) 62.4650% (K = 1) Class 1 ~ Class 9 denote Chromatin, Heterochromatin, Nuclear envelope, Nuclear matrix, Nuclear pore complex, Nuclear speckle, Nucleolus, Nucleoplasm and Nuclear PML body respectively. https://doi.org/10.1371/journal.pone.0195636.t005 Table 5. Prediction results for protein subnuclear of dataset 2. Class 1 ~ Class 9 denote Chromatin, Heterochromatin, Nuclear envelope, Nuclear matrix, Nuclear pore complex, Nuclear speckle, Nucleolus, Nucleoplasm and Nuclear PML body respectively. Class 1 ~ Class 9 denote Chromatin, Heterochromatin, Nuclear envelope, Nuclear matrix, Nuclear pore complex, Nuclear speckle, Nucleolus, Nucleoplasm and Nuclear PML body respectively. https://doi.org/10.1371/journal.pone.0195636.t005 https://doi.org/10.1371/journal.pone.0195636.t005 https://doi.org/10.1371/journal.pone.0195636.t005 and the results denote the proposed new feature expression CoPSSM still can resolve the imbalance problem of dataset 2. Subnuclear localization with new representation and kernel linear discriminant analysis Table 6. SE, SP, ACC and MCC for PseAAC, PSSM and the proposed CoPSSM on dataset 1. Type and Representation Evaluation Index SE SP ACC MCC Class 1 PseAAC 0.3023 0.9286 0.8050 0.2859 PSSM 0.5581 0.8964 0.8307 0.4565 The proposed CoPSSM 0.7093 0.9117 0.8747 0.5979 Class 2 PseAAC 0.3628 0.7908 0.6950 0.1492 PSSM 0.4159 0.7716 0.6957 0.1735 The proposed CoPSSM 0.5310 0.8756 0.8 0.4106 Class 3 PseAAC 0.2941 0.9774 0.9461 0.3088 PSSM 0 0.9866 0.9436 -0.0243 The proposed CoPSSM 0.2353 0.9831 0.9537 0.2696 Class 4 PseAAC 0.1667 0.9943 0.9538 0.2999 PSSM 0.2222 0.9918 0.9558 0.3382 The proposed CoPSSM 0.5 0.9805 0.9604 0.4939 Class 5 PseAAC 0.4167 0.9914 0.9723 0.4969 PSSM 0.5833 0.9863 0.9735 0.5697 The proposed CoPSSM 0.6667 0.9902 0.9810 0.6569 Class 6 PseAAC 0.32 0.9571 0.8775 0.3428 PSSM 0.3 0.9671 0.8867 0.3526 The proposed CoPSSM 0.26 0.9236 0.8548 0.1905 Class 7 PseAAC 0.8605 0.3590 0.6190 0.2550 PSSM 0.8163 0.4830 0.6583 0.3190 The proposed CoPSSM 0.7857 0.7269 0.7587 0.5135 Class 8 PseAAC 0 1 0.9213 - PSSM 0.1 0.9892 0.9223 0.1791 The proposed CoPSSM 0.2333 0.9485 0.9015 0.1847 Class 9 PseAAC 0.0541 0.9831 0.8954 0.0768 PSSM 0.1081 0.9945 0.9132 0.2447 The proposed CoPSSM 0.4324 0.9451 0.9035 0.3686 Class 10 PseAAC 0 0.9943 0.9616 -0.0137 PSSM 0 0.9973 0.9659 -0.0093 The proposed CoPSSM 0.25 0.9808 0.9604 0.2408 https://doi org/10 1371/journal pone 0195636 t006 e 6. SE, SP, ACC and MCC for PseAAC, PSSM and the proposed CoPSSM on dataset 1. 3, we can find that when K is equal to 9, 5 and 1, expressions PseAAC, PSSM and CoPSSM obtain their biggest prediction accuracy, respectively. For dataset 2, corresponding K values are 12, 3 and 1 separately. 3, we can find that when K is equal to 9, 5 and 1, expressions PseAAC, PSSM and CoPSSM obtain their biggest prediction accuracy, respectively. For dataset 2, corresponding K values are 12, 3 and 1 separately. The overall success rate (OSR) of PseAAC, PSSM and the proposed CoPSSM for different K values of KNN classifier Since various K values of the k-nearest-neighbour have an effect on the prediction perfor- mance, here what we displayed in Tables 4 and 5 are the highest OSR among different K val- ues. Therefore, we’ll provide following Figs 3 and 4 to show corresponding OSR of PseAAC, PSSM and the proposed CoPSSM when K ranges from 1 to 20. Figs 3 and 4 clearly display that the newly proposed feature representation CoPSSM per- forms the best, the often used 400-D PSSM is in second place and PseAAC is the last. From Fig PLOS ONE \| https://doi.org/10.1371/journal.pone.0195636 April 12, 2018 11 / 20 3, we can find that when K is equal to 9, 5 and 1, expressions PseAAC, PSSM and CoPSSM obtain their biggest prediction accuracy, respectively. For dataset 2, corresponding K values are 12, 3 and 1 separately. Subnuclear localization with new representation and kernel linear discriminant analysis Table 7. SE, SP, ACC and MCC for PseAAC, PSSM and the proposed CoPSSM on dataset 2. Type and Representation Evaluation Index SE SP ACC MCC Class 1 PseAAC 0.4848 0.7897 0.7324 0.2439 PSSM 0.6970 0.7445 0.7361 0.3579 The proposed CoPSSM 0.5960 0.9021 0.8447 0.4943 Class 2 PseAAC 0.2273 0.9896 0.9484 0.3335 PSSM 0.3636 0.9617 0.9318 0.3123 The proposed CoPSSM 0.3636 0.9648 0.9370 0.3151 Class 3 PseAAC 0.2459 0.9789 0.8773 0.3490 PSSM 0.2951 0.9561 0.8705 0.3174 The proposed CoPSSM 0.4918 0.9455 0.8902 0.4611 Class 4 PseAAC 0.1379 0.9922 0.9324 0.2576 PSSM 0.3103 0.9733 0.9297 0.3379 The proposed CoPSSM 0.4483 0.9752 0.9429 0.4629 Class 5 PseAAC 0.5570 0.8930 0.8355 0.4372 PSSM 0.6709 0.952 0.9031 0.6501 The proposed CoPSSM 0.7848 0.9576 0.9292 0.7424 Class 6 PseAAC 0.2985 0.9632 0.8635 0.3523 PSSM 0.4030 0.9341 0.8595 0.3697 The proposed CoPSSM 0.3284 0.9319 0.8544 0.2882 Class 7 PseAAC 0.8143 0.4533 0.6359 0.2874 PSSM 0.7134 0.6945 0.7045 0.4076 The proposed CoPSSM 0.7915 0.6767 0.7348 0.4716 Class 8 PseAAC 0 0.9897 0.9040 -0.03 PSSM 0.1622 0.9735 0.9071 0.1955 The proposed CoPSSM 0.1892 0.9543 0.8974 0.1634 Class 9 PseAAC 0 1 0.9674 - PSSM 0.0769 0.9951 0.9670 0.1482 The proposed CoPSSM 0.1538 0.9801 0.9571 0.1453 https://doi.org/10.1371/journal.pone.0195636.t007 Table 7. SE, SP, ACC and MCC for PseAAC, PSSM and the proposed CoPSSM on dataset 2. genetic algorithm (GA) to intelligently search the optimal kernel parameter. In the meantime, a new discriminant criterion was put forward to serve as fitness of the proposed optimization algorithm, dichotomous greedy genetic algorithm (DGGA). Hence, we named this method as CoPSSM with intelligent KLDA based on DGGA. Last, the dimension-reduced CoPSSM would be taken as input of KNN classifier to predict protein subnuclear location. genetic algorithm (GA) to intelligently search the optimal kernel parameter. In the meantime, a new discriminant criterion was put forward to serve as fitness of the proposed optimization algorithm, dichotomous greedy genetic algorithm (DGGA). Hence, we named this method as CoPSSM with intelligent KLDA based on DGGA. Last, the dimension-reduced CoPSSM would be taken as input of KNN classifier to predict protein subnuclear location. Here, the kernel linear discriminant analysis (KLDA) algorithm was implemented in MATLAB (R2011b version), using the famous Matlab Toolbox (developed by Laurens van der Maaten, Delft University of Technology). Prediction results of the proposed method: CoPSSM with intelligent KLDA based on DGGA Based on above analysis, we have proved that the newly proposed feature representation CoPSSM outperforms the two most frequently used representations, PseAAC and PSSM. Since an effective dimension reducing method played significant role in the prediction of pro- tein subnuclear location [4, 12, 13], here kernel linear discriminant analysis (KLDA) was applied to conduct dimensionality reduction on the proposed and best-performing feature expression CoPSSM. Kernel function of KLDA is the key point and it has a great influence on the final results. However, there is short of rational method to select the optimal kernel param- eter of kernel function at present. Inspired by it, we proposed a new algorithm based on the PLOS ONE \| https://doi.org/10.1371/journal.pone.0195636 April 12, 2018 12 / 20 PLOS ONE \| https://doi.org/10.1371/journal.pone.0195636 April 12, 2018 Different dimensionalities of the reduced data would influence the prediction results of protein subnuclear location, and since the Jackknife test method was very time-consuming, we took the defaulted value of KLDA as the reduced dimension with a view to the operating con- venience and conciseness. Namely, the number of protein type was set to the reduced dimen- sion. Hence the reduced dimensionality for datasets 1 and 2 were 10 and 9 respectively. Prediction results are as Table 8. From Table 8, we can clearly see that the overall success rates (OSR) for datasets 1 and 2 are 87.444% and 90.3361% respectively. Besides, prediction accuracy for each class is no less than 70% and even can reach up to 100% for Classes 3 and 10 on dataset 1. For dataset 2, there’s nar- rower fluctuation margin for each class. This is probably because the inherent attributes of 13 / 20 PLOS ONE \| https://doi.org/10.1371/journal.pone.0195636 April 12, 2018 Subnuclear localization with new representation and kernel linear discriminant analysis Fig 3. The OSR of PseAAC, PSSM and the proposed CoPSSM for different K values on dataset 1. https://doi.org/10.1371/journal.pone.0195636.g003 Fig 3. The OSR of PseAAC, PSSM and the proposed CoPSSM for different K values on dataset 1. https://doi org/10 1371/journal pone 0195636 g003 Fig 3. The OSR of PseAAC, PSSM and the proposed CoPSSM for different K values on dataset 1. htt //d i /10 1371/j l 0195636 003 Fig 3. The OSR of PseAAC, PSSM and the proposed CoPSSM for different K values on dataset 1. Fig 3. The OSR of PseAAC, PSSM and the proposed CoPSSM for different K values on dataset 1. https://doi.org/10.1371/journal.pone.0195636.g003 https://doi.org/10.1371/journal.pone.0195636.g003 different datasets lead to such a result. To show effectiveness of the proposed method, in the next section, it will be extended to predict protein subcellular localization. different datasets lead to such a result. To show effectiveness of the proposed method, in the next section, it will be extended to predict protein subcellular localization. PLOS ONE \| https://doi.org/10.1371/journal.pone.0195636 April 12, 2018 Experimental development: Predicting protein subcellular location via the proposed method, CoPSSM with intelligent KLDA based on DGGA To show the generalization of the proposed method, CoPSSM with intelligent KLDA based on DGGA, two protein subcellular benchmark datasets (datasets 3 and 4, shown in Table 3) were chosen to conduct the numerical experiment. Furthermore, dataset 4 was used as the real vali- dation set of dataset 3 instead of the Jackknife test; namely, as the training set, dataset 3 was tested by the Jackknife method while dataset 4 as the testing set was tested by the Independent method. Still, we firstly verified the newly proposed feature representation CoPSSM outper- forms the commonly used 40-D PseAAC and 400-D PSSSM. In the second place, the proposed method CoPSSM with intelligent KLDA based on DGGA was employed to predict protein subcellular location to demonstrate its effectiveness. The detailed experimental results are as Tables 9 and 10. From Table 9, we can get some useful information. For the Jackknife test dataset 3, it clearly indicates that the proposed new feature representation CoPSSM performs better than the com- monly used 40-D PseAAC and 400-D PSSM in discriminant ability. In addition, it shows CoPSSM can solve the data imbalance problem for Classes 3 and 4 to a certain degree as well. For the Independent test dataset 4, the performance of CoPSSM is superior to the 40-D PseAAC while is inferior to the 400-D PSSM; however, we still recommend the proposed CoPSSM in view of its lower dimensionality and better discriminant ability in dealing with the data imbalance problem than the 400-D PSSM. Despite all this, we can find that the proposed PLOS ONE \| https://doi.org/10.1371/journal.pone.0195636 April 12, 2018 14 / 20 Subnuclear localization with new representation and kernel linear discriminant analysis Fig 4. The OSR of PseAAC, PSSM and the proposed CoPSSM for different K values on dataset 2. https://doi.org/10.1371/journal.pone.0195636.g004 Fig 4. The OSR of PseAAC, PSSM and the proposed CoPSSM for different K values on dataset 2. https://doi.org/10.1371/journal.pone.0195636.g004 https://doi.org/10.1371/journal.pone.0195636.g004 CoPSSM still can’t predict proteins belonging to Class 6, which requires us to improve this fea- ture expression in the future work. Next, the proposed method CoPSSM with intelligent KLDA based on DGGA will be uti- lized to predict protein subcellular location. Numerical experiment results are shown in Table 10. https://doi.org/10.1371/journal.pone.0195636.t008 PLOS ONE \| https://doi.org/10.1371/journal.pone.0195636 April 12, 2018 Subnuclear localization with new representation and kernel linear discriminant analysis Table 9. Prediction results for protein subcellular location. Type Jackknife test for dataset 3 Independent test for dataset 4 PseAAC (40-D) PSSM (400-D) CoPSSM (210-D) PseAAC (40-D) PSSM (400-D) CoPSSM (210-D) Class 1 90.7895% 86.8421% 94.0790% 90.4762 89.0476 88.5714 Class 2 35.5263% 53.9474% 55.2632% 50 65 60 Class 3 0 8.3333% 50% 0 0 75 Class 4 0 83.3333% 83.3333% 100 100 100 Class 5 84.9462% 86.0215% 86.0215% 77.9710 79.1304 78.5507 Class 6 0 0 0 0 0 0 Class 7 60.1942% 54.3689% 61.1651% 46.1539 76.9231 84.6154 Class 8 48.2143% 65.1786% 53.5714% 61.2245 65.3061 57.1429 OSR 67.2282% (K = 5) 71.6692% (K = 8) 73.3538% (K = 6) 78.6936% (K = 6) 80.2488% (K = 12) 79.6268% (K = 5) Class 1 ~ Class 8 denote Cytoplasm, Extracell, Fimbrium, Flagellum, Inner membrane, Nucleoid, Outer membrane, Periplasm respectively. Table 9. Prediction results for protein subcellular location. the proposed method CoPSSM with intelligent KLDA based on DGGA, it’s necessary to com- pare it with state-of-the-art predictors on the same benchmark datasets. In the next section, detailed comparison results can justify efficiency of the proposed method in protein subnu- clear and subcellular localization. Experimental development: Predicting protein subcellular location via the proposed method, CoPSSM with intelligent KLDA based on DGGA From Table 10, we can clearly see that the overall success rates (OSR) for the training data- set 3 and the testing dataset 4 are 92.3430% and 94.7123% respectively, which proves the gener- alization of the proposed method in protein subcellular location. Last, to show effectiveness of Table 8. Prediction results of the proposed method. Table 8. Prediction results of the proposed method. Dataset 1 (reduced 10-D CoPSSM) Dataset 2 (reduced 9-D CoPSSM) Class 1 72.0930% 84.8485% Class 2 80.5310% 90.9091% Class 3 100% 86.8852% Class 4 94.4444% 89.6552% Class 5 83.3333% 88.6076% Class 6 98% 89.5522% Class 7 92.1769% 93.1596% Class 8 93.3333% 91.8919% Class 9 75.6757% 92.3077% Class 10 100% OSR 87.4440% (K = 13) 90.3361% (K = 10) https://doi.org/10.1371/journal.pone.0195636.t008 Table 8. Prediction results of the proposed method. Table 8. Prediction results of the proposed method. 15 / 20 PLOS ONE \| https://doi.org/10.1371/journal.pone.0195636 April 12, 2018 PLOS ONE \| https://doi.org/10.1371/journal.pone.0195636 April 12, 2018 15 / PLOS ONE \| https://doi.org/10.1371/journal.pone.0195636 April 12, 2018 https://doi.org/10.1371/journal.pone.0195636.t010 Subnuclear localization with new representation and kernel linear discriminant analysis Table 11. Comparison of overall success rate on the four benchmark datasets. Algorithm Representation and method OSR (%) Dataset 1 SubNucPred [33] SSLD and AAC based on SVM by Jackknife test 81.46 Effective Fusion Representations [4] DipPSSM with LDA based on KNN by 10-fold cross-validation 97 PseAAPSSM with LDA based on KNN by 10-fold cross-validation 84 The proposed method: CoPSSM with intelligent KLDA based on DGGA CoPSSM with KLDA based on KNN and Jackknife test 87.44 Dataset 2 Nuc-PLoc [7] Fusion of PsePSSM and PseAAC based on Ensemble classifier by Jackknife test 67.4 Effective Fusion Representations [4] DipPSSM with LDA based on KNN by 10-fold cross-validation 95.94 PseAAPSSM with LDA based on KNN by 10-fold cross-validation 88.1 The proposed method: CoPSSM with intelligent KLDA based on DGGA CoPSSM with KLDA based on KNN and Jackknife test 90.34 Dataset 3 Gneg-PLoc [34] Fusion of GO approach and PseAAC based on Ensemble classifier by Jackknife test 87.3 Nonlinear dimensionality reduction method [12] Fusion of PSSM and PseAAC with KLDA based on KNN by Jackknife test 98.77 The proposed method: CoPSSM with intelligent KLDA based on DGGA CoPSSM with KLDA based on KNN and Jackknife test 92.34 Dataset 4 Gneg-PLoc [34] Fusion of GO approach and PseAAC based on Ensemble classifier by Independent test 89.3 The proposed method: CoPSSM with intelligent KLDA based on DGGA CoPSSM with KLDA based on KNN and Independent test 94.71 https://doi.org/10.1371/journal.pone.0195636.t011 representation is less than that of information-rich fusion representation. For the extended experiment (predicting protein subcellular localization), results of dataset 3 and 4 still show the proposed method is effective. Besides, it proves again that the fusion representation con- tains more protein sequence information than the single feature expression, which makes the former get higher prediction result than the latter. Compare with existing prediction results Table 11 clearly shows comparison results of the overall success rate among the proposed method and different state-of-the-art algorithms on the four standard datasets. The most significant aspect that Table 11 reveals is that our proposed optimization algo- rithm can achieve good effect only with the single feature vector CoPSSM in the prediction of protein subnuclear and subcellular localization; whereas the other researches employed com- plex multiple representations, for instance, by fusing two kinds of different single representa- tions. For dataset 1, our proposed method outperforms the SubNucPred [33] and the second method in [4]. However, it is inferior to the first method in [4]. For dataset 2, the proposed method prevails over the Nuc-PLoc [7] and the second method in [4] while is still inferior to its first method. Note that the Jackknife test method used by us is more rigorous than the 10-fold cross-validation test employed in [4]. Hence, it reveals that our method is effective and meaningful, and at the same time, it also reveals that prediction efficiency of single feature Table 10. Prediction results of the proposed method for protein subcellular location. Type Jackknife test for dataset 3 Independent test for dataset 4 Reduced 8-D CoPSSM Reduced 8-D CoPSSM Class 1 136/152 = 89.4737% 186/210 = 88.5714% Class 2 67/76 = 88.1579% 18/20 = 90% Class 3 10/12 = 83.3333% 4/4 = 100% Class 4 6/6 = 100% 1/1 = 100% Class 5 181/186 = 97.3118% 339/345 = 98.2609% Class 6 6/6 = 100% 1/1 = 100% Class 7 97/103 = 94.1748% 12/13 = 92.3077% Class 8 100/112 = 89.2857% 48/49 = 97.9592% OSR 603/653 = 92.3430% (K = 13) 609/643 = 94.7123% (K = 5) https://doi.org/10.1371/journal.pone.0195636.t010 Table 10. Prediction results of the proposed method for protein subcellular location. PLOS ONE \| https://doi.org/10.1371/journal.pone.0195636 April 12, 2018 16 / 20 PLOS ONE \| https://doi.org/10.1371/journal.pone.0195636 April 12, 2018 PLOS ONE \| https://doi.org/10.1371/journal.pone.0195636 April 12, 2018 Author Contributions Author Contributions Conceptualization: Shunfang Wang, Yaoting Yue. Data curation: Yaoting Yue. Formal analysis: Shunfang Wang. Funding acquisition: Shunfang Wang. Investigation: Yaoting Yue. Methodology: Shunfang Wang, Yaoting Yue. Project administration: Shunfang Wang. Resources: Shunfang Wang. Software: Yaoting Yue. Supervision: Shunfang Wang. Validation: Shunfang Wang, Yaoting Yue. Visualization: Shunfang Wang, Yaoting Yue. Writing – original draft: Shunfang Wang, Yaoting Yue. Writing – review & editing: Shunfang Wang, Yaoting Yue. Data curation: Yaoting Yue. Formal analysis: Shunfang Wang. Funding acquisition: Shunfang Wang. Investigation: Yaoting Yue. Methodology: Shunfang Wang, Yaoting Yue. Project administration: Shunfang Wang. Resources: Shunfang Wang. Writing – original draft: Shunfang Wang, Yaoting Yue. Writing – original draft: Shunfang Wang, Yaoting Yue. Writing – review & editing: Shunfang Wang, Yaoting Yue. Writing – review & editing: Shunfang Wang, Yaoting Yue. Acknowledgments This research is supported by grants from National Natural Science Foundation of China (11661081) and Natural Science Foundation of Yunnan Province (2017FA032). Conclusions Until now, numerous studies have discussed protein subnuclear location [46–48]. Simulta- neously, prediction accuracy is getting higher and higher with newly developed methods and techniques [31, 49–52]. However, design complexity and time-consuming that come with it are still thorny problems that need to be addressed. Taking into these issues consideration, this paper tactfully put forward a new feature representation and idea to identify the protein sub- nuclear location. First, a more informative feature expression CoPSSM was created based on PSSM. Second, to search the bandwidth parameter of gauss kernel intelligently, we proposed an optimization algorithm DGGA based on GA. Third, to evaluate results of dimension reduc- tion, we proposed a new discriminant criterion as the fitness of DGGA. Compared with other people’s research findings, our method can get high performance partly. To verify generality and validity of the proposed method, two protein subnuclear standard datasets and Jackknife test, were considered to conduct the numerical experiments. Then, SE, SP, ACC and MCC were taken as the evaluation indexes. The experimental results were encouraging. Furthermore, the proposed method with Independent test was utilized to predict protein subcellular location and the obtained results still demonstrated its effectiveness. Whereas the dimensionality reduction algorithms KLDA undoubtedly adds the computational complexity to a certain degree, therefore, whether we can directly make good predictions with- out employing any dimensionality reduction algorithm deserves us to have thorough analysis and study in the future work. In addition, it remains an interesting challenge to obtain better Whereas the dimensionality reduction algorithms KLDA undoubtedly adds the computational complexity to a certain degree, therefore, whether we can directly make good predictions with- out employing any dimensionality reduction algorithm deserves us to have thorough analysis and study in the future work. In addition, it remains an interesting challenge to obtain better PLOS ONE \| https://doi.org/10.1371/journal.pone.0195636 April 12, 2018 17 / 20 Subnuclear localization with new representation and kernel linear discriminant analysis representations for protein subnuclear localization and to study other machine learning classi- fication algorithms [4]. representations for protein subnuclear localization and to study other machine learning classi- fication algorithms [4]. 7. Shen HB, Chou KC. Nuc-PLoc: a new web-server for predicting protein subnuclear localization by fus- ing PseAA composition and PsePSSM. 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Bio Systems. 2016 Dec: 37–45. https:// doi.org/10.1016/j.biosystems.2015.12.002 PMID: 26724384 20 / 20 PLOS ONE \| https://doi.org/10.1371/journal.pone.0195636 April 12, 2018
https://openalex.org/W3198627427	https://books.fe.up.pt/index.php/feup/catalog/download/978-972-752-279-8/978-972-752-279-8_0056-0064/269?inline=1	English	null	Work-related musculoskeletal disorder and its costs: a short review	null	2,021	cc-by	4,642	Abstract Introduction: Musculoskeletal disorder can result from extreme physical demands at work. It has risen, mainly due to high muscle demands and static postures and generates a significant economic burden to the companies. This short review aims to point out the statistics of the most affected body parts by work- related musculoskeletal disorders, assess absenteeism and presenteeism costs, and evaluate the effectiveness of the interventions. Methodology: According to the PRISMA Statement, the search was performed in 5 electronic databases (Scopus, Web of Science, Science Direct, Pubmed). Articles selection was made by the title and abstract analysis, especially those aiming to explain and validate the subject. If the abstract fulfils the objective, the articles were read, and studies were considered that met the defined eligibility criteria. Were chosen articles that realised measurements or considered interventions in healthy human beings, especially in the working population, dated from 2015 to 2020. Results and Discussion: The scrutinised articles were conclusive that the most affected part of the body are the upper limbs, the neck and the upper back. The statistics of the affected workers varied according to the country and analysed tasks, but they converge concerning the most affected body parts. It could be noted that women are more affected by musculoskeletal disorders than men, especially older women; the reason, however, is not clearly explained. Muscle injury can lead high economic burden, mostly due to absenteeism and presenteeism. It was noted that the expenditure due to presenteeism is higher comparing to absenteeism in both developed and emergent countries. Conclusions: Due to high costs, companies are deploying strategies to improve work conditions and aware the workers about health and safety. Interventions have shown to be effective in reducing the risk of injuries. Work-related musculoskeletal disorders can be extremely damaging to the workers’ health and are costly to the companies. Support supervisor interventions effectively improve work conditions and reduce the risk of damaging outcomes, increasing workers’ health, work ability, and, consequently, productivity. Keywords: Injury, Absenteeism, Presenteeism, Strategies. Work-related musculoskeletal disorder and its costs: a short review Ana Sophia Rosado1, J. Santos Baptista2 and J. C. Guedes3 1Faculty of Engineering, University of Porto, PT (anasgmr@yahoo.com.br) ORCID 0000-0002-4935-4171, 2Associated Laboratory for Energy, Transports, and Aeronautics - LAETA (PROA), Faculty of Engineering, University of Porto, PT (jsbap@fe.up.pt) ORCID 0000-0002-8524-5503, 3Associated Laboratory for Energy, Transports, and Aeronautics - LAETA (PROA), Faculty of Engineering, University of Porto, PT (jccg@fe.up.pt) ORCID 0000-0003-2367-2187. https://doi.org/10.24840/978-972-752-279-8 0056-0064 METHODOLOGY The review was done by the analysis of articles, which could be reviews and individual researches. The analysis of each article was verified, on the first moment, the title and the abstract. If the abstract fulfils the goal, then the lecture of the full articles was performed. The selection reflected only articles that considered healthy human beings and workers of working age. Articles evaluating outdoor work and measurement methods utilising Rula, Reba, Ocra, Owas, and NIOSH were not considered. The databases used were: Scopus, Web of Science, Science Direct, Pubmed. The words used in the investigation were: - cost absenteeism AND employers; - increase health AND absenteeism; - loss productivity AND injury; - musculoskeletal AND absenteeism AND presenteeism AND cost; - musculoskeletal AND body region; - musculoskeletal disease AND body part; - musculoskeletal disorder AND reduce productivity; - work capacity AND tiredness; - work capacity AND increase AND health; - work capacity evaluation AND tiredness; - work productivity AND absenteeism AND presenteeism. This systematic review was performed according to the PRISMA Statement (Moher D. et al., 2009), and the research was updated to March 2021. The investigation of the articles resulted in: summary of the collected articles with 33 209 articles pre-selected. Applying the exclusion criteria, 24 648 papers were excluded by “date” (articles published in the period between 2015 and 2020 were included); 3 446 were rejected by “document type” (only articles and review articles were accepted); 11 texts were refused due to “language” (only English was accepted). After articles exclusion according to the explained criteria, were read the titles and abstracts of the remaining ones. 5 073 articles were excluded because they are not consistent with the goal of this Short review. Finally, 31 articles were used for analysis in this work. Of all analysed articles, thirteen exhibited the statistics of the most injured part of the body by WMSD, five articles compared the costs of absenteeism and presenteeism due to musculoskeletal disorder, and the other investigated articles justified this systematic review. Articles before 2015 were considered due to their importance about the topic. INTRODUCTION The high physical load and repetitive movements are the leading causes of musculoskeletal disorders associated with emotional stress (Atroszko et al., 2020). Physical exhaustion results in muscular frailty and can be aggravated by awkward postures, repetitive tasks, and static position, which propitiate the risk of injury (Hembecker et al., 2017) (Knoop et al., 2019). Work-related musculoskeletal disorders (WMSD) are a reality in companies worldwide and represent the highest disease rate (Acaröz et al., 2019). It is a crucial factor to be assessed since it directly relates to the employees’ health, safety, and productivity (Caldwell et al., 2019). Illnesses originated from overwork precedes high economic burden to workers companies, especially due to presenteeism and absenteeism (Silva et al., 2018). Musculoskeletal disorder is the leading cause of work absenteeism in Europe, affecting over 40 million workers. It leads to disability around the globe in 20%-33% and represents up to 40% of occupational costs (Moussavi, Zare, Mahdjoub, & Grunder, 2019) (Santos & Mendes, 2020) (Crawford et al., 2020). The World Health Organization (WHO) recognises that an unsuitable working environment precedes physical and mental health problems, raising the risk of sick leave and loss of productivity (Atroszko et al., 2020). Considering the high associated costs, companies are becoming aware of improving working conditions, as well as reducing productivity losses and sick leave (Jonge et al., 2019) (Atroszko et al., 2020). Although various factors contribute to the loss of work capacity, WMSD has proved to be one of the most important. This study is a short review to point out the most affected body parts and 56 evaluate absenteeism and presenteeism as harm outcomes resulting from musculoskeletal disorders. evaluate absenteeism and presenteeism as harm outcomes resulting from musculoskelet disorders. Rosado A. S. et al., 2021. Work-related musculoskeletal disorder and its costs: a short review. Most common sites pain Work-related musculoskeletal disorders can affect workers and companies of different sectors. The consequences can be damaging to the health of the workers and the economy of companies (Acaröz et al., 2019). Due to its severity, several kinds of research have been made to understand the most affected gender, regions of the body, and costs. It was noted that women are more susceptible to WMSD, and it is twice as common in older women than in man (Pensola et al., 2016; Knoop et al., 2019; Acaröz et al., 2019; Troelstra et al., 2020). However, no author has explained clearly the reason why women are most affected. 57 Rosado A. S. et al., 2021. Work-related musculoskeletal disorder and its costs: a short review. Several researchers have scrutinised the part of the body with the highest rate of WMSD. Figure 1 shows the result of thirteen articles (number 1-13 on the X-axis). It is known that musculoskeletal injuries disturb workers from different sectors. Tasks with physical demands and intellectual workers are both touched by bodily injuries. Figure 2 illustrates the result of thirteen analysed articles (numbers 1-13 on the X-axis), concerning the most affected body parts, considering different jobs, such as health workers (nurses, physicians, physical therapists), financial services workers, manufacturing workers, academicians, supermarket cashiers, construction workers. It must be highlighted that the used methods for the evaluation mainly were the Nordic Musculoskeletal Questionnaire, but also were used the Dutch Musculoskeletal Questionnaire, Work Ability Index, Institute for Medical Technology Assessment Productivity Cost Questionnaire (iPCQ), Work Productivity and Activity Impairment Questionnaire, and health examination. In addition, musculoskeletal pain and disorder from the past twelve months were considered. Figure 1, illustrates the most affected parts of the body due to WMSD, in indoor activities. Figure 1, illustrates the most affected parts of the body due to WMSD, in indoor activities. g , p y , Figure 1. The parts of the body most affected by WMSD. Figure 1. The parts of the body most affected by WMSD. The evaluation of the articles permits to conclude that the incidence of WMSD is mainly in the neck and back. Figure 2 illustrates the statistics of the three most affected body parts. It must be underlined that the back pain can be related to the upper back or lower back. Some of the articles did not explain the exact location. 58 Rosado A. S. et al., 2021. Work-related musculoskeletal disorder and its costs: a short review. Figure 2. The three most affected body parts by WMSD. Figure 2. The three most affected body parts by WMSD. The discrepancies between the statistics results might be attributed to the characteristics of the evaluated population and the work characteristics (Ezzatvar et al., 2020), but there is a consensus on the most affected body parts. In this sense, according to the European Agency for Safety and Health at Work, lower back pain is one of the main causes of disability and sick leave (Algarni, Alkhaldi, et al., 2020; Acaröz et al., 2019). Rosado A. S. et al., 2021. Work-related musculoskeletal disorder and its costs: a short review. The severity of WMSD may increase the risk of disability, chronic pain, sick leave, reduced work capability, and early retirement (Miranda et al., 2010; Phongamwong & Deema, 2015; Hallman et al., 2019; Troelstra et al., 2020; Vieira & Sato, 2020). Costs It is estimated that an economic burden due to WMSD in Europe is approximately 2% of the Gross Domestic Product (GDP) (Bevan, 2015; Svendsen et al., 2020; Santos & Mendes, 2020; Crawford et al., 2020). The monetary expenses are mainly due to presenteeism and absenteeism (Nagata et al., 2018). Absenteeism can be easily calculated since it represents sick leave. However, evaluating the cost of presenteeism is more complicated because the loss of productivity is often a hidden cost. It reduces the effectiveness due to pain or disorders (Ammendolia et al., 2016), which usually offers unseen expenses. Figure 3 reflects the economic burden (percentage) due to presenteeism and absenteeism in five developed and developing countries to illustrate the importance of reducing musculoskeletal disorder. 59 59 Rosado A. S. et al., 2021. Work-related musculoskeletal disorder and its costs: a short review. Figure 3. Comparing the total cost of absenteeism and presenteeism in five countries. Figure 3. Comparing the total cost of absenteeism and presenteeism in five countries. Figure 3. Comparing the total cost of absenteeism and presenteeism in five countries. Figure 3. Comparing the total cost of absenteeism and presenteeism in five countries. It has been assumed that presenteeism costs exceed the costs of absenteeism (van der Burg et al., 2020; Lohaus & Habermann, 2019). Figure 3 makes it clear that independently of developed and emergent countries, the costs due to sick leave and loss of productivity are high. It can be understood that human beings are equal concerning the body’s movements and adoption of postures. Workers not affected by WMSD symptoms have a higher quality of life (Algarni, Alkhaldi, et al., 2020), which is reflected in positive outcomes and efficiency at work and in private life. It demonstrates that improvements at work, such as awareness of adopting correct postures and reducing stressors, can minimise harmful outcomes (Acaröz et al., 2019). Rosado A. S. et al., 2021. Work-related musculoskeletal disorder and its costs: a short review. Strategies Companies have a great challenge to protect workers’ health and ensure a safe workplace (Boschman et al., 2015). To improve work conditions and minimise the incidence of WMSD, occupational and hygiene professionals should implement different strategies (Nordstoga et al., 2019). Well-planned policies can increase workers’ health and reduce unnecessary costs (Crawford et al., 2020). Interventions, such as mapping the process considering individual risk factors and productivity demands, are proven efficient to achieve excellent outcomes (Atroszko et al., 2020). Training is also a low-cost and effective measure to minimise hidden productivity losses (Brunner et al., 2019), sickness- absence, and consequently prolong workers careers (Nielsen & Midtsundstad, 2020). Prevent harm conditions at work is crucial to avoid absenteeism and economic losses, as well as the injuries caused by the muscular response to excessive overwork (McDonald, Mulla, & Keir, 2019). Tactics with supportive supervisors have been demonstrated to be effective to improve work conditions and reduce the risk of damaging consequences (Baral et al., 2018; Brunner et al., 2019; Söderbacka et al., 2020). However, planning an effective strategy, risk management, and the best strategy to be taken must be evaluated according to the reality and feasibility of each organisation (Ammendolia et al., 2016; Crawford et al., 2020). CONCLUSIONS Work-related musculoskeletal disorders reflect a high economic burden to companies around the world, as well as prejudice worker health. The consequences to the individuals can reflect lost productivity and, in most serious cases, sickness absence. The more significant cost is due to presenteeism, more than absenteeism, although it is difficult to evaluate its cost since it is a hidden cost. It is known that developed and underdeveloped countries have significant losses, which could be minimised with effective and inexpensive strategies. It is important to aware workers about physical health, especially taking care of the neck and back, which are the most affected body parts. Well-designed strategies are essential to improve work conditions and should be taking according to the reality experienced. Algarni, F. S., Alkhaldi, H. A., Zafar, H., Kachanathu, S. J., Al-Shenqiti, A. M., & Altowaijri, A. M. (2020). Self-Reported Musculoskeletal Disorders and Quality of Life in Supermarket Cashiers. International Journal of Environmental Research and Public Health, 17(24), 1–14. https://doi.org/10.3390/ijerph17249256 Algarni, F. S., Kachanathu, S. J., & Alabdulwahab, S. S. (2020). A Cross-Sectional Study on the Association of Patterns and Physical Risk Factors with Musculoskeletal Disorders among Academicians in Saudi Arabia. BioMed Research International. https://doi.org/10.1155/2020/8930968 Causes Strategies Companies have a great challenge to protect workers’ health and ensure a safe workplace (Boschman et al., 2015). To improve work conditions and minimise the incidence of WMSD occupational and hygiene professionals should implement different strategies (Nordstoga et al. 2019). Well-planned policies can increase workers’ health and reduce unnecessary costs (Crawford e al., 2020). Interventions, such as mapping the process considering individual risk factors and productivity demands, are proven efficient to achieve excellent outcomes (Atroszko et al., 2020) Training is also a low-cost and effective measure to minimise hidden productivity losses (Brunne et al., 2019), sickness- absence, and consequently prolong workers careers (Nielsen & Midtsundstad, 2020). Prevent harm conditions at work is crucial to avoid absenteeism and economic losses, as well a the injuries caused by the muscular response to excessive overwork (McDonald, Mulla, & Keir 2019). Tactics with supportive supervisors have been demonstrated to be effective to improve work conditions and reduce the risk of damaging consequences (Baral et al., 2018; Brunner e al., 2019; Söderbacka et al., 2020). However, planning an effective strategy, risk management, and the best strategy to be taken must be evaluated according to the reality and feasibility of each organisation (Ammendolia e al., 2016; Crawford et al., 2020). CONCLUSIONS Work-related musculoskeletal disorders reflect a high economic burden to companies around It must be emphasised that workers have been affected by multisite pain. The most common combination is neck and back pain (Pensola et al., 2016). Musculoskeletal disorder in several parts of the body dramatically reduces work ability (Bayattork et al., 2019; Hallman et al., 2019; Ezzatvar et al., 2020), with detrimental effects on workers and companies. Causes Professionals from different sectors are negatively affected by overload, improper ergonomic conditions, and a lack of attention to the principles of biomechanics (Ameer & Ashour, 2020). It can be said that the musculoskeletal system is primarily affected by repetitive work and overload, but static postures also can be damaging to physical health, increasing the risk of WMSD (Hembecker et al., 2017; Algarni et al., 2020). It must be highlighted that extreme demanding tasks are related to psychological stress, directly associated with functional stress and illnesses (Karnaukh & Shevtsova, 1991; Moussavi, Zare, Mahdjoub, & Grunder, 2019). Besides the high demand at work, sedentary behaviour and unhealthy eating habits are also detrimental to health. Regarding physical activity (PA), it has been settled that leisure PA promotes health and decreases in 20% the risk of long-term sickness absence. Contrary to leisure PA, a high occupational PA increases the risk of long-term sickness absence (Gupta et al., 2020). Muscular injuries are caused by the insufficient restoration of energy and strength. In its turn, the first symptom of WMSD is usually muscular pain, which is directly associated with reduced work capacity, loss of productivity, and in most severe cases it can lead to absence due to illness (Jonge et al., 2019; Hallman et al., 2019; Ameer & Ashour, 2020; Skovlund et al., 2020). Other serious consequences of WMSD are turnover intentions, depression (Frone & Blais, 2019), and disability retirement (Pensola et al., 2016). Emphasising this sentence, in Finland, the most common reason for early retirement is musculoskeletal problems (33%) (Söderbacka et al., 2020). These harmful results have a direct impact on workers’ health and high costs. 60 Rosado A. S. et al., 2021. Work-related musculoskeletal disorder and its costs: a short review. It must be emphasised that workers have been affected by multisite pain. The most common combination is neck and back pain (Pensola et al., 2016). Musculoskeletal disorder in several parts of the body dramatically reduces work ability (Bayattork et al., 2019; Hallman et al., 2019; Ezzatvar et al., 2020), with detrimental effects on workers and companies. It must be emphasised that workers have been affected by multisite pain. The most common combination is neck and back pain (Pensola et al., 2016). Musculoskeletal disorder in severa parts of the body dramatically reduces work ability (Bayattork et al., 2019; Hallman et al., 2019 Ezzatvar et al., 2020), with detrimental effects on workers and companies. Egypt. Physiotherapy Quarterly, 28(4), 42–4 . https://doi.org/10.5114/PQ.2020.96235 Egypt. Physiotherapy Quarterly, 28(4), 42–4 . https://doi.org/10.5114/PQ.2020.96235 Ammendolia, C., Côté, P., Cancelliere, C., Cassidy, J. D., Hartvigsen, J., Boyle, E., … Iii, B. A. (2016). 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M. de A., & Sato, T. de O. (2020). Prevalence of multisite pain and association with work ability – Cross-sectional study. Musculoskeletal Science and Practice, 50. https://doi.org/10.1016/j.msksp.2020.102279 complaints : a prospective cohort study. RMD Open - Rheumatic & Musculoskeletal Diseases, 6(2), 1–8. https://doi.org/10.1136/rmdopen-2020-001281 Vieira, L. M. S. M. de A., & Sato, T. de O. (2020). Prevalence of multisite pain and association with work ability – Cross-sectional study. Musculoskeletal Science and Practice, 50. https://doi.org/10.1016/j.msksp.2020.102279 complaints : a prospective cohort study. RMD Open - Rheumatic & Musculoskeletal Diseases, 6(2), 1–8 https://doi.org/10.1136/rmdopen-2020-001281 https://doi.org/10.1136/rmdopen-2020-001281 Vieira, L. M. S. M. de A., & Sato, T. de O. (2020). Prevalence of multisite pain and association with work ability – Cross-sectional study. Musculoskeletal Science and Practice, 50. https://doi.org/10.1016/j.msksp.2020.102279 Vieira, L. M. S. M. de A., & Sato, T. de O. (2020). Prevalence of multisite pain and association with work ability – Cross-sectional study. Musculoskeletal Science and Practice, 50. https://doi.org/10.1016/j.msksp.2020.102279 64 64 Rosado A. S. et al., 2021. Work-related musculoskeletal disorder and its costs: a short review.
https://openalex.org/W4309066411	https://commonplace.knowledgefutures.org/pub/j0a8lxly/download/pdf	English	null	Designing for Emergent Workflow Cultures: eLife, PRC, and Kotahi	Commonplace	2,022	cc-by	4,716	ABSTRACT Scholarly publishing is evolving, and there is a need to understand and design the new (emergent) workflows while also designing technology to capture and support these processes. This article documents an ongoing collaboration to develop technology to meet emergent workflows in scholarly publishing, namely Publish- Review-Curate (PRC). We explore this topic with different eLife1 PRC community stakeholders using Kotahi,2 a flexible open-source scholarly publishing platform that can support variant workflows (built by Coko3). Commonplace • Series 2.2 Community-led Editorial Management Designing for Emergent Workow Cultures: eLife, PRC, and Kotahi Commonplace • Series 2.2 Community-led Editorial Management Designing for Emergent Workow Cultures: eLife, PRC, and Kotahi Commonplace • Series 2.2 Community-led Editorial Management Designing for Emergent Workow Cultures: eLife, PRC, and Kotahi Designing for Emergent Workow Cultures: eLife, PRC, and Kotahi Designing for Emergent Workow Cultures: eLife, PRC, and Kotahi What is PRC? A preprint is a manuscript that has been shared publicly before submission to a scholarly journal. Preprints have been a popular pre-publication process for many years, however recently there has been an explosion of this kind of early sharing of research due to COVID-19. There is a need to sort ‘the good’ preprints from ‘the bad’ so researchers can take advantage of early research without being mired in a torrent of questionable results. Consequently, volunteer communities of researchers have emerged to review preprints. This activity has been particularly popular during the COVID-19 era. There is a need to sort ‘the good’ preprints from ‘the bad’ so researchers can take advantage of early research without being mired in a torrent of questionable results. Consequently, volunteer communities of researchers have emerged to review preprints. This activity has been particularly popular during the COVID-19 era. These communities publish their reviews of preprints to assist other researchers in finding and interpreting the research. These communities publish their reviews of preprints to assist other researchers in finding and interpreting the research. To illustrate this approach simply, we can represent preprint review processes with the initials PR (Publish, Review): 1. Publish - researchers publish preprints on ‘preprint servers’ 1. Publish - researchers publish preprints on ‘preprint servers’ 2. Review - researchers review the preprints (and share the reviews) 2. Review - researchers review the preprints (and share the reviews) Traditional journal publishing, on the other hand, could be described with the initials RP (Revie Traditional journal publishing, on the other hand, could be described with the initials RP (Review, Publish): 1 R i h i b itt d i t 1. Review - researchers review a submitted manuscript 2. Publish - the manuscript is published 1. Review - researchers review a submitted manuscript 2. Publish - the manuscript is published This simple flip of the PR vs RP highlights the fundamental difference in approach. Preprint review processes flip the typical path of a manuscript through a journal on its head. This simple flip of the PR vs RP highlights the fundamental difference in approach. Preprint review processes flip the typical path of a manuscript through a journal on its head. PRC adds a new component to this dynamic: Curation. PRC adds a new component to this dynamic: Curation. 1. Publish - researchers publish preprints on ‘preprint servers’ 2. What is PRC? Each of these stakeholders are simultaneously working out their own workflow and how to work together. play an important foundational role. Each of these stakeholders are simultaneously working out their own workflow and how to work together. Journal workflows are far easier to design technical platforms for as the workflows are ‘known’ and journals ‘own’ the process from end-to-end. There is no ‘established’ PRC workflow as there is for traditional journals and we have multiple interacting stakeholders simultaneously working out ‘how this works.’ This makes working within the PRC domain very exciting as there is a lot to work through. eLife has recently started down this path of discovery by transforming its own publishing model to a Publish- Review-Curate (PRC) workflow.7 Workflow transformation of this kind is radical — it requires the transformation of the cultural and technical operating environment of not just its own journal but the entire ecosystem in which it resides. However, eLife is in a good position to do this as recent internal analysis shows that in 2021 over 80% of papers under review at eLife were already available on bioRxiv, medRxiv or arXiv,8 that is “the eLife community has always strongly supported preprinting.”9 Sciety as an enabler for PRC While operating under a PRC model for their own publishing operations, eLife also is encouraging and facilitating others to do the same. To this end, eLife invested in building Sciety,10 a website that helps aggregate all of the preprint review happening across the web with additional tools for curation of reviews. Sciety started as a simple aggregator giving PRC organisations a place to showcase their evaluation activities (e.g. those occurring in Kotahi), and for some, providing a home on the web to describe who they are and what they are doing. Sciety quickly evolved into a place that also enabled the curation of reviews by organisations11 and individual researchers.12 These features of Sciety, together with Sciety’s commitment to the Principles of Open Scholarly Infrastructure,13 make it a key partner to Kotahi to assist any organization to review and publicize their preprint evaluation activities. What is PRC? Review - researchers review the preprints (and share the reviews) 3. Curate - a journal curates preprints from already published and reviewed sources. Michael Eisen et al formulate this as ‘Publication as curation.’4 3. Curate - a journal curates preprints from already published and reviewed sources. Michael Eisen et al formulate this as ‘Publication as curation.’4 2 Commonplace • Series 2.2 Community-led Editorial Management Designing for Emergent Workow Cultures: eLife, PRC, and Kotahi The top diagram is a journal, followed by preprint review, and Publish-Review-Curate The top diagram is a journal, followed by preprint review, and Publish-Review-Curate PRC is effectively a hybrid approach combining community-evolved preprint review processes and traditional effectively a hybrid approach combining community-evolved preprint review processes and tra processes. The preprint and community review processes assist with the early discovery and interpretation of research, while journal titles are maintained in this ecosystem as they are important “...for many researchers as they pursue their careers.”5 Journals also act as a secondary mechanism for improving the communication of the results and building trust via careful curation and recommendations. many researchers as they pursue their careers.”5 Journals also act as a secondary mechanism for improving the communication of the results and building trust via careful curation and recommendations. many researchers as they pursue their careers.”5 Journals also act as a secondary mechanism for improving the communication of the results and building trust via careful curation and recommendations. In this evolving domain, the journals are not discarded but find a new emphasis, as Stern and O’Shea identified : In this evolving domain, the journals are not discarded but find a new emphasis, as Stern and O’Shea identified : “As high-volume publishing platforms continue to grow, we’ll need curation services that select articles of interest for specific target audiences. Today’s selective journals and scientific societies could be well positioned to provide such services.”6 “As high-volume publishing platforms continue to grow, we’ll need curation services that select articles of interest for specific target audiences. Today’s selective journals and scientific societies could be well positioned to provide such services.”6 Designing technology for PRC is challenging as the territory is, in effect, a fusion of multiple emergent workflow cultures including preprint review communities, ‘journals-that-curate,’ and preprint servers which 3 Commonplace • Series 2.2 Community-led Editorial Management Designing for Emergent Workow Cultures: eLife, PRC, and Kotahi play an important foundational role. 4. Use and Review Using the features and evaluating them. Using the features and evaluating them. The following sections examine in brief detail each of the above stages. The following sections examine in brief detail each of the above stages. Design and Build Process To aid us in the process of designing workflow for multiple preprint review communities, we decided to use the Workflow Sprint14 methodology. “A Workflow Sprint is a fast and efficient methodology for helping organizations understand and optimize their publishing workflows before making technology choices.”15 4 Commonplace • Series 2.2 Community-led Editorial Management Designing for Emergent Workow Cultures: eLife, PRC, and Kotahi Designing for Emergent Workow Cultures: eLife, PRC, and Kotahi 1. Operational stakeholders are best placed to describe and optimize workflow 2. Group discussion simultaneously expedites the process and adds clarity l k We leveraged this existing framework and further adapted the methodology to cater for the f multiple individual stakeholders, each with multiple team members multiple time zones Our aim has been to work with multiple preprint review communities in parallel to co-design their workflow and build features to encapsulate these ideas within Kotahi. We held Workflow Sprints with each preprint review community individually so they could focus on their specific workflow. We kept sessions to one hour, forcing us to focus and ‘get to the point.’ Workflow Sprints were followed by build processes. In its entirety, the process looked something like this: 5 Commonplace • Series 2.2 Community-led Editorial Management Designing for Emergent Workow Cultures: eLife, PRC, and Kotahi The 4 Steps from a Workflow Sprint to Build and back again The 4 Steps from a Workflow Sprint to Build and back again 1. Workflow Sprint A series of regular remote meetings in rapid succession to design and optimize the workflow vision of the group, followed by: A series of regular remote meetings in rapid succession to design and optimize the workflow vision of the group, followed by: 2. Feature Design A series of sessions to design the technology (extensions to Kotahi) that supports the workflow. These sessions focused on extending Kotahi to meet the functional requirements of the workflow. 3. Build Building the features. Step 1. Workflow Sprint The fundamental principle of this part of the process is to focus on the communities workflow (how they do things) and not technology. These discussions investigated what was done now and how it might be done better. Documentation of the outcomes were via: 6 Commonplace • Series 2.2 Community-led Editorial Management Designing for Emergent Workow Cultures: eLife, PRC, and Kotahi 1. Workflow notation in the form of WFMU16 2. Some basic illustrative flow diagrams 2. Some basic illustrative flow diagrams Workflow Sprint meetings were facilitated by Adam Hyde and Ryan Dix-Peek from Coko. Workflow Sprint meetings were facilitated by Adam Hyde and Ryan Dix-Peek from Coko. Asset created after a Workflow Sprint Asset created after a Workflow Sprint Step 3. Build After a solution (generally a Kotahi feature of fixed scope) was agreed Ryan would create documentation for the Coko17 developers so they could start programming. Step 4. Use and Review Once the feature(s) have been delivered, the preprint review stakeholders then use the features in production. Feedback is then presented back to The Kotahi Project Manager, and the process to improve the workflow and technology would begin again. Step 2. Feature Design Workflow Sprints inevitably generate many feature ideas to solve some of the workflow problems. In particular, eLife’s Paul Shannon and Coko’s Adam Hyde would often have creative suggestions for solutions, having seen innumerable existing solutions in varying contexts. Sometimes participants with no history of platform design, would offer better solutions. In each case, these ideas were considered by the entire group and discussed. Kotahi Project Manager Ryan Dix-Peek would document the suggested solutions and begin feature designs. These designs were then brought back to the group for discussion, improvement, and validation. Commonplace • Series 2.2 Community-led Editorial Management Designing for Emergent Workow Cultures: eLife, PRC, and Kotahi Mocks from Kotahi Feature Design for a Threaded Discussion feature Mocks from Kotahi Feature Design for a Threaded Discussion feature Participants The participants of each session included: The participants of each session included: Lessons learned We have learned an immense amount about the PRC ecosystem and how to build technologies to support nascent workflows. The following are some top-level takeaways in brief detail. Facilitator (Coko staff: Adam Hyde or Ryan Dix-Peek) Stakeholders from eLife/Sciety (usually Paul Shannon) Stakeholders from the PRC organization Facilitator (Coko staff: Adam Hyde or Ryan Dix-Peek) Stakeholders from eLife/Sciety (usually Paul Shannon) Stakeholders from the PRC organization Meeting technologies used For these remote meetings, we used minimal technologies (mostly BigBlueButton18 and Miro19) and focused on discussion with documentation of outcomes often following the event. 8 Commonplace • Series 2.2 Community-led Editorial Management Designing for Emergent Workow Cultures: eLife, PRC, and Kotahi Diagrams used to help clarify Workflow when working remotely Diagrams used to help clarify Workflow when working remotely Most documentation resided in shared online word processor docs and shared collaborative boards (such as the Miro example above). Between meetings, Kotahi Project Manager Ryan Dix-Peek would spend time creating visuals as documentation to help present the conclusions back in the follow-on meetings for validation. Workflow MarkUp (WFMU) and ‘block level’ system diagrams have both been proven to be useful tools. Lessons learned We have learned an immense amount about the PRC ecosystem and how to build technologies to support nascent workflows. The following are some top-level takeaways in brief detail. 9 Commonplace • Series 2.2 Community-led Editorial Management Designing for Emergent Workow Cultures: eLife, PRC, and Kotahi Designing for Emergent Workow Cultures: eLife, PRC, and Kotahi 1 . No two PRC Workflows look the same. We learned a lot from working through PRC workflows with multiple communities. As expected, with an emergent field, there are varying visions, and there are multiple places in the workflow where there is some notable divergence in approach (with brief notes and lessons learned): Triage Some selection processes are very hierarchical and others exhibit a flatter dynamic more consistent with ‘open’ communities. Many preprint review triage processes look very much like a standard journal triage process, with an academic board reviewing content and deciding what should be reviewed. In other communities, preprints are ‘self-selected’ by reviewers to review with no prior triage process. Reviewer selection Some communities self-select preprints to review, others invite reviewers. Still others are a hybrid of both approaches. Some preprint communities suffer the same problems as journals when it comes to identifying and chasing possible reviewers. Submission/ingestion process By submit, we mean the manual ‘submission of an existing preprint for review’. By ingest, we mean automatic processes that import preprint metadata from preprint servers for review. It is interesting to note that PRC ‘submission’ or ingestion processes seldom require the actual preprint (PDF) itself to reside within the reviewing platform, instead preferring to link to the preprint manuscript on the hosting preprint server via DOI. In this way, preprint review processes differ from journal processes, and to some degree (caveat below), this can simplify submission/ingestion while at the same time also offers many challenges for this emergent sector moving forward (see below). Approaches to submission/ingestion have been disparate—some review communities automatically ingest periodically (eg daily/on demand) numerous preprints simultaneously from existing preprint servers; some communities prefer authors to volunteer (submit) their preprint for review, others will reach out to authors and invite them to submit. Still, others submit on behalf of the authors and invite the author to validate the submission before reviewing. Automatic ingestion processes do sometimes prove problematic since there is no standardization of data available through preprint server APIs. Consequently, if a preprint review community sources preprints from multiple preprint servers, there is no guarantee the required metadata profile can be obtained. Since most preprint communities work within a specific, narrow research domain, the ingestion process also needs to target specific categories of research, and this data/search mechanism is not always available to the required fidelity through the existing APIs. We have, in some cases, needed to use AI services (such as Semantic Scholar20) for matching keywords to research domains and then cross-referencing to other services such as EPMC21 or preprint servers. This can be technically complex. Additionally, since preprint reviews are necessarily time-sensitive, ingestion of content needs to be date filtered and we have, in some circumstances, needed to cross reference various services to determine the ‘submission age’ of the preprint. Interestingly, this lack of standardization of metadata available from preprint servers has forced some communities to ask authors to ‘resubmit’ a preprint to a specific, preferred server to ensure consistency of available data. 10 10 Commonplace • Series 2.2 Community-led Editorial Management Designing for Emergent Workow Cultures: eLife, PRC, and Kotahi Review process Review processes can vary wildly. Some communities require one round of reviews only, others look all the world like a typical journal review process, including the expectation that authors will consider the reviews and update the preprint (on the host preprint server). Interestingly, this is where the entire PRC flow (review community --> journal) sometimes becomes out of sync. A preprint might be undergoing multiple review rounds with a preprint community when a journal accepts the manuscript. In this scenario, the authors may consider the journal process has a higher priority and abandon participating in the preprint review process. The format of the review also varies considerably between communities. Some communities treat reviews separately, others prefer a collaborative review process. Some communities actively engage the author in a threaded discussion and curate that discussion as part of a shared (published) author response. Author involvement How the author is engaged (if at all), has been an area of large divergence. As alluded to above, some communities establish a relationship with the author early by inviting the author to submit or ‘self-nominate’ their preprint for review. Other communities do not engage the author at all, and still others formulate the review process with multiple rounds requiring the author to engage deeply with the process. This area has possibly been one of the most interesting as it often points towards internal cultural values. In some cases, there is a lot of concern about maintaining a ‘respectful’ relationship with the author by involving them in the review process. In other cases, the expediency of reviews is considered more important and the author may not be aware that their preprint is under review at all. 3. Preprint Servers are increasingly becoming a center of gravity It has been clear throughout this process that there is a lot of reverence from the researcher community towards the various preprint servers which service their domain. Preprint servers are the technical, social and content foundations upon which PRC is built, and consequently, preprint review communities and journals-that-curate are both reliant on the ongoing good faith and technical viability of the preprint server teams. The question going forward is whether the preprint servers will be able to meet and maintain the growing requirements of the emergent PRC processes. In particular, issues of metadata standardization, process synchronization, and external service integrations (incoming and outgoing APIs) are critical for the advancement of this sector. 2. The arc of the PR universe sometimes bends towards journal workflows 2. The arc of the PR universe sometimes bends towards journal workflows It has at times been interesting to hear preprint communities start layering on features incrementally and, as a whole, the workflow starts looking more and more like a typical journal workflow. This has been fascinating to see as sometimes PR communities are antagonistic to the value metrics and inefficiencies they believe the current journal ecosystem supports. It has, at times, been necessary to challenge some preprint stakeholders on their priorities. Are they trying to discard/change/improve the current way of doing things, or are they layering legacy journal processes on top of preprint servers? Publishing There is a lot of variance here. The format of the published data varies immensely from community to community. The content can have any mix of metadata, commentary, author responses, and ‘endorsements’. 11 11 Commonplace • Series 2.2 Community-led Editorial Management Designing for Emergent Workow Cultures: eLife, PRC, and Kotahi Some share a synopsis of the preprint with a review or recommendation to a community homepage. Others publish to shared spaces (such as Sciety). 4. Software is a conversation We have built features into Kotahi for each workflow requirement which has required a lot of conversation and co-design. Building a scholarly publishing platform that can manage this high degree of process variance is not the simplest of tasks. We have achieved support for all of these disparate workflows, primarily because of good faith, open discussion, and the co-design principles of Workflow Sprints. We have achieved support for all of these disparate workflows, primarily because of good faith, open discussion, and the co-design principles of Workflow Sprints. We have often found that a feature that initially seemed like a large variance with current features turns out, after many hours of drilling down into the problem together, to require only a small extension to the platform. If we were too quick to design and build, or we abandoned the principles of co-design, we would have many complex features in Kotahi that ‘approximated the solution’ and did not actually solve the problem. Thankfully this hasn’t been the case. It is also true that sometimes the preprint stakeholders have a high-level idea of what they are trying to achieve, but when we drill down, this reveals contradictions or consequences they find difficult to accept. This has led to many interesting workflow, technical, and philosophical discussions about what the community is trying to achieve. 12 Commonplace • Series 2.2 Community-led Editorial Management Designing for Emergent Workow Cultures: eLife, PRC, and Kotahi At other times, we have needed to build in abstractions to allow for configurable options when workflows are clearly divergent and require new features. Thankfully, the Workflow Sprint design and build processes have lent themselves well to meeting the needs of each community’s individual visions of PRC workflow. The process has allowed us to front-load discussion and discovery in an expedient, collaborative manner. It would be a mistake to adopt a development process that designs too far ahead when it comes to features that support disparate emergent workflows. 5. PR, RP, and PRC can all coexist in the same platform One of the design philosophies of Kotahi is that all submit-review-publish processes look the same from a ‘bird’s eye view’, and it is possible to build a system that can support a ‘domain agnostic’ approach. From a certain distance, for example, funding review looks very similar to both journal workflows and preprint community review workflows: 1. Submit 2. Review 3. Improve 4. Share Kotahi was originally designed as a journal system through a series of Workflow Sprints with the journal Collabra: Psychology.22 eLife recognized that the ‘bird’s eye view’ design philosophy could work for supporting PRC workflows, and the collaboration began. These experiences have really tested the ability for a single system to support a domain-agnostic approach (above) and we are happy to validate this approach so far. Kotahi can support an enormous amount of variance in Journal and PRC workflows. With Kotahi quickly becoming the platform of choice for PRC communities, and with a growing number of organizations (notably publishing service providers) using Kotahi for journals, we have a lot of faith in the Workflow Sprint processes and the Kotahi platform to fulfill and support these disparate needs going forward. Summary Designing scholarly technology to support emergent workflows is initially a conversational process that leads to shared concepts, documented (speculative) requirements, and an evolving shared language. When these foundational elements are, at least partly, established, the process can begin to incrementally move forward with initial technical designs. The technologies built from these designs, in turn, lead to ‘real-world’ experiences which feed back into the conversations, and we witness the gradual but progressive discarding and replacement of legacy ideas, The technologies built from these designs, in turn, lead to ‘real-world’ experiences which feed back into the conversations, and we witness the gradual but progressive discarding and replacement of legacy ideas, 13 13 Commonplace • Series 2.2 Community-led Editorial Management Designing for Emergent Workow Cultures: eLife, PRC, and Kotahi Designing for Emergent Workow Cultures: eLife, PRC, and Kotahi language, and processes. One of the challenges with this process is designing and building a technical platform that can cater for all these divergent workflow pathways within the one system. This has required a lot of careful technical design decisions by the Coko23 development team. The pressure of these real world disparate requirements is welcomed and has benefited the platforms architecture and utility immensely. PRC is a series of connected and emergent workflows with no fixed approach. This presents many challenges for platform development intended to encapsulate these processes. The eLife-Coko collaboration has explored this in depth through the design and build of features to support a large variety of PRC workflows in Kotahi. Our findings essentially validate that it is important to front load the workflow co-design process before building features and testing in the real world. We have found that patient discussion and co-design have been the main attributes of our success in building a platform through which these communities can collaborate to expedite the sharing of reviews and research. Footnotes 1. https://https://elifesciences.org/ ↩ 2. https://kotahi.community ↩ 3. https://coko.foundation ↩ 4. Michael B Eisen, Anna Akhmanova, Timothy E Behrens, Diane M Harper, Detlef Weigel, Mone Zaidi (2020) Peer Review: Implementing a "publish, then review" model of publishing eLife 9:e64910 https://doi.org/10.7554/eLife.64910 ↩ 5. Michael B Eisen, Anna Akhmanova, Timothy E Behrens, Diane M Harper, Detlef Weigel, Mone Zaidi (2020) Peer Review: Implementing a "publish, then review" model of publishing eLife 9:e64910 https://doi.org/10.7554/eLife.64910 ↩ 6. Stern BM, O’Shea EK (2019) A proposal for the future of scientific publishing in the life sciences. PLoS Biol 17(2): e3000116. https://doi.org/10.1371/journal.pbio.3000116 ↩ 7. https://elifesciences.org/inside-elife/54d63486/elife-s-new-model-changing-the-way-you-share-your- research ↩ 8. https://elifesciences.org/inside-elife/e5f8f1f7/elife-latest-what-we-have-learned-about-preprints ↩ 9. https://elifesciences.org/inside-elife/00f2f185/elife-latest-preprints-and-peer-review ↩ 10. https://sciety.org/ ↩ 1. https://https://elifesciences.org/ ↩ 2. https://kotahi.community ↩ 3. https://coko.foundation ↩ 4. Michael B Eisen, Anna Akhmanova, Timothy E Behrens, Diane M Harper, Detlef Weigel, Mone Zaidi (2020) Peer Review: Implementing a "publish, then review" model of publishing eLife 9:e64910 https://doi.org/10.7554/eLife.64910 ↩ 5. Michael B Eisen, Anna Akhmanova, Timothy E Behrens, Diane M Harper, Detlef Weigel, Mone Zaidi (2020) Peer Review: Implementing a "publish, then review" model of publishing eLife 9:e64910 https://doi.org/10.7554/eLife.64910 ↩ 6. Stern BM, O’Shea EK (2019) A proposal for the future of scientific publishing in the life sciences. PLoS Biol 17(2): e3000116. https://doi.org/10.1371/journal.pbio.3000116 ↩ 7. https://elifesciences.org/inside-elife/54d63486/elife-s-new-model-changing-the-way-you-share-your- research ↩ 8. https://elifesciences.org/inside-elife/e5f8f1f7/elife-latest-what-we-have-learned-about-preprints ↩ 9. https://elifesciences.org/inside-elife/00f2f185/elife-latest-preprints-and-peer-review ↩ 10. https://sciety.org/ ↩ 1. https://https://elifesciences.org/ ↩ 2. https://kotahi.community ↩ 3. https://coko.foundation ↩ 4. Michael B Eisen, Anna Akhmanova, Timothy E Behrens, Diane M Harper, Detlef Weigel, Mone Zaidi (2020) Peer Review: Implementing a "publish, then review" model of publishing eLife 9:e64910 https://doi.org/10.7554/eLife.64910 ↩ (2020) Peer Review: Implementing a "publish, then review" model of publishing eLife 9:e64910 https://doi.org/10.7554/eLife.64910 ↩ 5. Michael B Eisen, Anna Akhmanova, Timothy E Behrens, Diane M Harper, Detlef Weigel, Mone Zaidi (2020) Peer Review: Implementing a "publish, then review" model of publishing eLife 9:e64910 https://doi.org/10.7554/eLife.64910 ↩ 6. Stern BM, O’Shea EK (2019) A proposal for the future of scientific publishing in the life sciences. PLoS Biol 17(2): e3000116. https://doi.org/10.1371/journal.pbio.3000116 ↩ 7. https://elifesciences.org/inside-elife/54d63486/elife-s-new-model-changing-the-way-you-share-your- research ↩ 8. https://elifesciences.org/inside-elife/e5f8f1f7/elife-latest-what-we-have-learned-about-preprints ↩ 9. https://elifesciences.org/inside-elife/00f2f185/elife-latest-preprints-and-peer-review ↩ 10. https://sciety.org/ ↩ 5. Michael B Eisen, Anna Akhmanova, Timothy E Behrens, Diane M Harper, Detlef Weigel, Mone Zaidi (2020) Peer Review: Implementing a "publish, then review" model of publishing eLife 9:e64910 https://doi.org/10.7554/eLife.64910 ↩ 6. Stern BM, O’Shea EK (2019) A proposal for the future of scientific publishing in the life sciences. PLoS Biol 17(2): e3000116. https://doi.org/10.1371/journal.pbio.3000116 ↩ 7. Footnotes https://elifesciences.org/inside-elife/54d63486/elife-s-new-model-changing-the-way-you-share-your- research ↩ 14 Commonplace • Series 2.2 Community-led Editorial Management Designing for Emergent Workow Cultures: eLife, PRC, and Ko 11. https://sciety.org/groups/biophysics-colab/lists ↩ 12. ↩ 13. https://blog.sciety.org/open-scholarly-infrastructure/ ↩ 14. https://coko.foundation/articles/understanding-and-implementing-workflow-first-design-workflow- sprints.html ↩ 15. https://coko.foundation/articles/understanding-and-implementing-workflow-first-design-workflow- sprints.html ↩ 16. https://coko.foundation/articles/understanding-and-implementing-workflow-first-design-workflow- sprints.html ↩ 17. https://coko.foundation ↩ 18. https://bigbluebutton.org/ ↩ 19. https://miro.com/ ↩ 20. https://www.semanticscholar.org/ ↩ 21. https://europepmc.org/ ↩ 22. https://online.ucpress.edu/collabra ↩ 23. https://coko.foundation ↩ Commonplace • Series 2.2 Community-led Editorial Management Designing for Emergent Workow Cultures: eLife, PRC, and Kota 11. https://sciety.org/groups/biophysics-colab/lists ↩ 12. ↩ 13. https://blog.sciety.org/open-scholarly-infrastructure/ ↩ 14. https://coko.foundation/articles/understanding-and-implementing-workflow-first-design-workflow- sprints.html ↩ 15. https://coko.foundation/articles/understanding-and-implementing-workflow-first-design-workflow- sprints.html ↩ 16. https://coko.foundation/articles/understanding-and-implementing-workflow-first-design-workflow- sprints.html ↩ 17. https://coko.foundation ↩ 18. https://bigbluebutton.org/ ↩ 19. https://miro.com/ ↩ 20. https://www.semanticscholar.org/ ↩ 21. https://europepmc.org/ ↩ 22. https://online.ucpress.edu/collabra ↩ 23. https://coko.foundation ↩ Commonplace • Series 2.2 Community-led Editorial Management Designing for Emergent Workow Cultures: eLife, PRC, and Kotahi 15
https://openalex.org/W2946846380	https://www.hal.inserm.fr/inserm-02295791/file/s12889-019-7031-7.pdf	English	null	A cross sectional survey to estimate prevalence and associated factors of asthma on Reunion Island, Indian Ocean	BMC public health	2,019	cc-by	7,333	To cite this version: Jean-Louis Solet, Chantal Raherison-Semjen, Éric Mariotti, Yann Le Strat, Anne Gallay, et al.. A cross sectional survey to estimate prevalence and associated factors of asthma on Reunion Island, Indian Ocean. BMC Public Health, 2019, 19 (1), pp.663. 10.1186/s12889-019-7031-7. inserm-02295791 A cross sectional survey to estimate prevalence and associated factors of asthma on Reunion Island, Indian Ocean A cross sectional survey to estimate prevalence and associated factors of asthma on Reunion Island, Indian Ocean Jean-Louis Solet, Chantal Raherison-Semjen, Éric Mariotti, Yann Le Strat, Anne Gallay, Éric Bertrand, Nishrod Jahaly, Laurent Filleul Jean-Louis Solet, Chantal Raherison-Semjen, Éric Mariotti, Yann Le Strat, Anne Gallay, Éric Bertrand, Nishrod Jahaly, Laurent Filleul © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Abstract Background: Previous studies on asthma mortality and hospitalizations in Reunion Island indicate that this French territory is particularly affected by this pathology. Epidemiological studies conducted in schools also show higher prevalence rates in Reunion than in Mainland France. However, no estimates are provided on the prevalence of asthma among adults. In 2016, a cross-sectional survey was conducted to estimate the prevalence of asthma and to identify its associated factors in the adult population of Reunion Island. Methods: A random sample of 2419 individuals, aged 18–44 years, was interviewed by telephone using a standardized, nationally validated questionnaire. Information was collected on the respiratory symptoms, description of asthma attacks and triggering factors for declared asthmatics, as well as data on the indoor and outdoor home environment. “Current asthma” was defined as an individual declaring, at the time of the survey, having already suffered from asthma at some point during his/her life, whose asthma was confirmed by a doctor, and who had experienced an asthma attack in the last 12 months or had been treated for asthma in the last 12 months. “Current suspected asthma” was defined as an individual presenting, in the 12 months preceding the study, groups of symptoms suggestive of asthma consistent with the literature. Results: The estimated prevalence of asthma was 5.4% [4.3–6.5]. After adjustment, women, obesity, a family member with asthma, tenure in current residence and presence of indoor home heating were associated with asthma. The prevalence of symptoms suggestive of asthma was 12.0% [10.2–13.8]. After adjustment, marital status, passive smoking, use of insecticide sprays, presence of mold in the home and external sources of atmospheric nuisance were associated with the prevalence of suspected asthma. Conclusion: Preventive actions including asthma diagnosis, promotion of individual measures to reduce risk exposure as well as the development of study to improve knowledge on indoor air allergens are recommended. Keywords: Asthma, Epidemiology, Prevalence, Reunion, Indian Ocean A cross sectional survey to estimate prevalence and associated factors of asthma on Reunion Island, Indian Ocean J-L. Solet1* , C. Raherison-Semjen2, E. Mariotti3, Y. Le Strat4, A. Gallay4, E. Bertrand5, N. Jahaly6 and L. Filleul1 HAL Id: inserm-02295791 https://inserm.hal.science/inserm-02295791v1 Submitted on 24 Sep 2019 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. Solet et al. BMC Public Health (2019) 19:663 https://doi.org/10.1186/s12889-019-7031-7 Background French overseas departments exceed the national aver- age [3]. Reunion, a French overseas department, an is- land in the Indian Ocean with an area of 2512-km2 and a population of 850,996 (2016 population estimate based on the French National Institute of Statistics and Eco- nomic Studies (INSEE) census). Previous studies have indicated that the area is particularly affected by asthma. Adjusting for age and gender, asthma mortality rates are 3 to 5 times higher than in mainland France and hospi- talisations for asthma are twice as common [4–6]. School-based studies have shown that Reunion is one of The World Health Organisation (WHO) estimates that 235 million people around the world have asthma and that it led to 255,000 deaths in 2005 [1]. In Europe, the prevalence of asthma decreases from north to south. The United Kingdom and Ireland are particularly af- fected, with prevalences surpassing 30%. France falls into the middle of the range [2]. However, prevalences in the * Correspondence: jean-louis.solet@santepubliquefrance.fr 1Santé Publique France [The French Public Health Agency], Indian Ocean Regional Office, Saint-Denis, Reunion, France Full list of author information is available at the end of the article * Correspondence: jean-louis.solet@santepubliquefrance.fr 1Santé Publique France [The French Public Health Agency], Indian Ocean Regional Office, Saint-Denis, Reunion, France Full list of author information is available at the end of the article Solet et al. BMC Public Health (2019) 19:663 Page 2 of 9 Page 2 of 9 the regions with the highest prevalences among both young children and adolescents [7, 8]. recorded, and a random draw undertaken to select the person to be interviewed. The questionnaires were ad- ministered in French and Creole by trained investigators, using ASKIA software, and the CATI (computer-assisted telephone interview) system. The questionnaire included questions about sociodemographics (e.g. gender, age, commune of residence, level of education, and place of birth), anthropometrics (e.g. height, weight, and body mass index (BMI)), smoking habits, chronic health prob- lems and diseases, symptoms suggestive of asthma, asthma attacks and trigger factors, and the interviewees’ home environment (both interior and exterior). In order to reduce the reporting bias, asthma was not mentioned at the beginning of the interview. Investigators intro- duced the questionnaire as a general survey on health and the home environment. All questions relating to symptoms were asked prior to the investigators men- tioning asthma itself. However, information on the prevalence of asthma among the general adult population has been lacking. For this reason, a cross sectional survey was conducted in 2016 to estimate the prevalence of asthma in the gen- eral population of adults aged between 18 and 44 years living in Reunion. The secondary objectives of the study were to describe the characteristics of asthmatic patients and of individuals with symptoms suggestive of asthma, and to study how the domestic environment affects the risk of asthma in order to improve prevention strategies, particularly individual-level measures. Study population Between 8th February and 1st June 2016 a phone survey was conducted amongst a random and representative sample of individuals aged between 18 and 44 years. People under the age of 18 were excluded because there was already data available on asthma in children and ad- olescents in Reunion [7, 8]. People over 44 years were excluded in order to reduce the risk of misclassification of asthma symptoms as those of chronic obstructive pul- monary disease (COPD), which is more common among older smokers. The questionnaire primarily included questions from the European Community Respiratory Health Survey (ECRHS) [9] and the Epidemiological study on the Genetics and Environment of Asthma (EGEA) [10]. The questionnaire was developed in con- junction with respiratory and allergy medical specialists in Reunion. Case definitions C h - Current asthma: any person reporting ever receiving a diagnosis of asthma from a doctor, and who had experi- enced an asthma attack or was treated for asthma in the preceding 12 months. - Suspected current asthma: Any person reporting one or more groups of symptoms suggestive of asthma con- sistent with the literature in the preceding 12 months (wheezing, resting, nocturnal and exertion dyspnea, noc- turnal respiratory discomfort, nocturnal cough) [12] but not classified as asthmatic according to the definition of current asthma. Data analysis Th i The target population was stratified into two groups. The first group consisted of individuals who resided in households equipped with a landline telephone, while the second group was composed of individuals residing in households equipped with a mobile phone but no landline telephone (the “only mobile” group). The pro- portion of interviewees from each group (70% from the landline group and 30% from the only mobile group) was determined based on reference statistics [11]. Sam- pling involved dialling randomly generated telephone numbers in order to include people not listed in the phone book for the landline group and clients from the different local mobile operators for the mobile phone group. Telephone numbers that did not lead to an inter- view (e.g. due to non-response, refusal, unavailability, in- accessibility, non-eligibility) were replaced by successive incrementing. The replacement of an unsuccessful num- ber due to non-response only occurred after repeated calls, set at a minimum of 8 attempts, at different times of the day and days of the week. The estimators produced were consistent with the sam- pling design (stratification, sampling weights, primary sampling units). Additionally, post-stratification weight- ing was implemented, using the “Reweight Iterative Method”, to adjust estimates to the population age and gender distribution obtained from the INSEE 2013 cen- sus of the population. These estimates were calculated along with their 95% confidence intervals (CI) taking into account the adjustment made to the sample and its impact on increasing the variance of the proportion esti- mator. Chi-square tests were used to compare propor- tions between groups. Univariate analysis was used to calculate odds ratios and their 95% CI estimated by the Woolf method. Data were analysed using Xlstat and R software. Multi- variate analysis using logistic regression models was undertaken to identify the factors associated with current asthma and suspected asthma. In each model, a pre-selection of explanatory variables was conducted by reviewing all variables considered as risk factors for asthma (e.g. gender, age, smoking, BMI, In responding households with more than one eligible household member, the household composition was Page 3 of 9 Page 3 of 9 Page 3 of 9 Solet et al. BMC Public Health (2019) 19:663 Solet et al. BMC Public Health (2019) 19:663 Page 3 of 9 having a family member with asthma) as well as vari- ables that could potentially be associated with asthma (such as the home environment). Results The final sample consisted of 2419 individuals, including 1706 individuals in the “landline” group and 713 individ- uals in the “only mobile” group. Achieving these re- quired more than 4700 call hours and more than 208, 000 telephone calls (including false numbers, non- responses, ineligibility, refusals). A total of 6939 inter- views were started, i.e. the phone was answered and in- formation on household composition collected. The cessation of 4338 interviews was mainly due to two rea- sons: the absence of persons aged 18–44 in the house- hold (2855 cases) and the selection of a participant not available at the time of the call (1271 cases). The inter- view was ceased due to refusal to participate in 212 calls. In addition, 182 interviews were ceased during the inter- view following the participant’s refusal to continue. These incomplete questionnaires were not included in the analysis. Amongst valid questionnaires, the response rate to individual questions was high (greater than 95%). Throughout the survey, quality checks of the interviews were regularly carried out by the supervisor responsible for monitoring the study. The results of the multivariate analysis adjusted by age are shown in Table 3. After adjustment, the prevalence of asthma was higher among women than men and among people who re- ported having family members with asthma and those living in homes with heating. However, the prevalence decreased with length of time in the current home. Ethics At the time of the survey, French regulations did not require the approval of an ethics committee for ob- servational studies that did not include performing acts or using products on the participants, as was the case for this study. The study protocol was approved by an internal expert committee (CCEP) of the French Public Health Agency. In addition, a declar- ation was made to the National Commission on In- formatics and Freedoms (CNIL) in accordance with the Data Protection Act. Contacted households and selected participants were informed that participation was optional and about their rights of access, rectification and opposition to the data concerning them. Regarding domestic environmental factors, univariate analyses showed that the prevalence of asthma was higher amongst those living in their current residence for less than 5 years (OR: 3.56; 95% CI: 1.77–7.64). The prevalence was significantly higher when there was a re- ported source of moisture in immediate proximity to the residence (less than 10 m) (OR: 1.71; 95% CI: 1.18–2.47). Similarly, having obstructed air vents in the home was associated with a higher prevalence (OR: 3.95; 95% CI: 1.72–9.06). The presence of mould, moist spots, or fun- gus in the home was associated with a higher prevalence of asthma (OR: 1.54; 95% CI: 1.08–2.19). The permanent presence of cockroaches (defined as sightings every, or almost every day) was also associated with a higher prevalence (OR: 2.33; 95% CI: 1.27–4.28). Prevalence of current asthma The estimated prevalence of current asthma was 5.4% [4.3–6.5] among the population of individuals aged be- tween 18 and 44 years living in Reunion (Table 2). The prevalence was higher in women (7.0%; 95% CI: 2.2–4.8) than in men (3.5%; 95% CI: 5.4–8.6) (p < 0.0001) across all age groups. Women aged between 18 and 24 years were most affected, with a prevalence of 9.8% (95% CI: 5.8–13.8). The prevalence of current asthma did not vary signifi- cantly according to the occupational status, socio- professional category, level of education, place of birth and/or the number of years the person had lived on Reunion Island, or the place of employment. Obesity (defined as a BMI greater than or equal to 30 kg/m2) was related to asthma, with a prevalence of 8.3% (95% CI: 4.7–11.9) versus 5.4% (95% CI: 4.0–6.8) among people with a normal body mass (a BMI of 18.5 to 24.9 kg/m2). (Odd Ratio (OR): 1.59; 95% CI: 1.00–2.52). Those with an immediate family member diagnosed with asthma had a higher prevalence of asthma of 8.3% (95% CI: 5.9–10.1) compared to a prevalence rate of 3.6% for those without a family history (95% CI: 2.5–4.7), OR: 2.33 (95% CI: 1.62–3.35). Data analysis Th i At the end, 23 ex- planatory variables were chosen to be tested in each model. The glmulti library in the R software was then used for an initial selection of variables through the like- lihood ratio test and the Akaike information criterion. The first order interactions were then added to the ini- tial models and an exhaustive selection of models was conducted with the glumult algorithm. Prevalence of current asthma Prevalence of suspected asthma The prevalence of persons who reported experiencing the symptoms suggestive of asthma in the last 12 months was estimated to be of 12.0% (95% CI: 10.2–13.8%). Among these, 8.8% did not meet the definition of current asthma. This is important because it suggests that a large percentage of people with symptoms The average age of participants was 34 years old [18 to 44 years old]. The sample was comprised of 47.3% men and 52.7% women, with a gender ratio of 0.81 (Table 1). Solet et al. BMC Public Health (2019) 19:663 Page 4 of 9 Table 1 Sociodemographic profile of respondents, Reunion, 2016 N observed % estimated 95% CI Gender Male 1080 47.3% [45.0%; 49.6%] Female 1339 52.7% [50.4%; 55.0%] Age groups 18–19 years 138 8.3% [7.0%; 9.6%] 20–24 years 401 18.1% [16.3%; 19.9%] 25–29 years 301 17.3% [15.5%; 19.1%] 30–34 years 369 17.2% [15.4%; 19.0%] 35–39 years 460 18.7% [16.9%; 20.5%] 40–44 years 750 20.4% [18.5%; 22.3%] Occupation-status Actively employed (salaried or otherwise) 1271 48.4% [46.1%; 50.7%] Actively unemployed 521 22.7% [20.7%; 24.7%] Never worked (inactive) 179 9.4% [8.0%; 10.8%] Other inactive (retirees…) 448 19.5% [17.6%; 21.4%] Socio-professional categories Clerks 747 29.6% [27.5%; 31.7%] Laborers 419 18.6% [16.8%; 20.4%] Intermediate occupations 368 13.7% [12,1%; 15,3%] Senior managers, liberal professions 170 5.6% [4.5%; 6.7%] Artisans, traders, company managers 64 2.6% [27.5%; 31.7%] Farmers 24 1.0% [0.5%; 1.5%] Others and inactives 627 28.8% [26.7%; 30.9%] Marital status Single 1256 56.2% [53.9%; 58.5%] Married or civil partnership 674 23.1% [21.1%; 25.1%] Not married but cohabiting 394 17.9% [16.1%; 19.7%] Divorced 74 2.2% [1.5%; 2.9%] Widower 10 0.3% [0.0%; 0.6%] Unspecified 11 0.5% [0.2%; 0.8%] Place of birth Reunion 1914 79.6% [77.7%; 81.5%] Mainland France 370 14.4% [12.8%; 16.0%] Mayotte 39 2.5% [1.8%; 3.2%] Madagascar 38 1.4% [0.9%; 1.9%] Mauritius 19 0.6% [0.2%; 1.0%] Other 39 1.5% [0.9%; 2.1%] Length of time in Reunion Less than 5 years 153 7.0% [5.8%; 8.2%] Between 5 and 10 years 107 4.4% [3.4%; 5.4%] Between 10 and 15 years 138 4.5% [3.5%; 5.5%] Table 1 Sociodemographic profile of respondents, Reunion, 2016 (Continued) N observed % estimated 95% CI Between 15 and 30 years 895 43.8% [41.5%; 46.1%] More than 30 years 1125 40.2% [37.9%; 42.5%] Unspecified 1 0.0% – Total 2419 100% Table 1 Sociodemographic profile of respondents, Reunion, 2016 (Continued) suggestive of asthma have not been diagnosed. Prevalence of suspected asthma In addition, 2.2% of those who met the definition of current asthma did not experience any symptoms suggestive of asthma in the last 12 months. Overall, 14.2% (95% CI: 12.3–16.1) of all people aged between 18 and 44 years reported having current asthma or the symptoms sug- gestive of asthma. The prevalence of suspected current asthma was slightly higher in women (9.7%; 95% CI: 7.8–11.6) than in men (7.9%; 95% CI: 6.0–9.8), though the difference was not statistically significant (p = 0.120). Univariate analysis showed that the prevalence of suspected current asthma did not vary significantly according to occupa- tional status, socio-professional category, level of edu- cation, place of birth and/or the number of years the person has been living in Reunion, or the place of residence or employment. The main risk factors for suspected asthma were obesity (OR: 1.52; 95% CI: 1.02–2.28), being underweight (OR: 1.76; 95% CI: 1.07–2.92), having an immediate family member with asthma (OR: 1.70; 95% CI: 1.28–2.26), being around regular smokers (OR: 1.34; 95% CI: 1.01–1.78), and marital status, with a higher prevalence amongst sin- gle, divorced, or widowed persons than for those who were married, in a civil union, or cohabitating (OR: 1.73; 95% CI: 1.27–2.36). The results of univariate analyses on environmental factors showed that the presence of mould, moisture, or fungi in the home was associated with a higher preva- lence of suspected asthma: 12.0% (95% CI: 9.4–14.6) ver- sus 7.9% (95% CI: 6.3–9.5), (p = 0.0015), (OR: 1.58; 95% CI: 1.18–2.10). Similarly, the presence of cockroaches was associated with a higher prevalence of suspected asthma, with the prevalence increasing with the fre- quency of sightings (Table 4). Regular use (defined as every day or almost every day) of insecticide sprays within the residence was also asso- ciated with a higher prevalence of suspected asthma: 22.8% (95% CI: 12.6–33.0), (p < 0.0001), OR: 3.22 (95% CI: 1.86–5.55) compared to those not using sprays regu- larly: 8.6% (95% CI: 6.3–10.9). The results of the multivariate analysis are shown in Table 5. Solet et al. Discussion The prevalence of current asthma amongst 18 to 44 years olds in Reunion, estimated at 5.4% (95% CI: 4.3– 6.5), is similar to the previously published prevalence in mainland France of 6.0% (95% CI: 5.7–6.4) amongst in- dividuals over 15 years in a 2003 national health survey [2] and 7.1% amongst those aged over 15 years in the 2012 Institute for Research and Documentation in Health Economics (IRDES) study [13]. Other studies show clear differences in the prevalence of asthma in young children between the two territories [2, 3, 7, 8]. This result raises the hypothesis that the percentage of children on Reunion Island whose asthma has resolved by adolescence is greater than in mainland France. This possibility merits further examination through comple- mentary studies. We found a higher prevalence of asthma in women than in men, as well as a correlation with obesity, results which have been frequently described in the literature [9, 15–18]. The higher prevalence of asthma among people with a family member with asthma (8.0% versus 3.6%) is consistent with our current knowledge and un- derstanding of the role of genetic factors in the develop- ment of asthma [19]. However, the case definition for current asthma used in this survey was more restrictive than those used in other national studies since, in addition to an asthma at- tack and/or treatment during the last 12 months, it in- cluded a diagnosis of asthma by a doctor and the criteria of having previously suffered from asthma during the person’s life. Therefore the prevalence in Reunion might have been underestimated due to the more specific case definition. The association between the asthma and the number of years the person has been living in his/her current residence is more complex to understand. The possibil- ity of new furniture being a source of allergens or irri- tants was suggested. Univariate analysis indicated an association between the presence of moist spots and mould inside the home and the prevalence of asthma (though not a statistically significant factor in the logistic regression model). Many studies have found that traces of moisture in the home are linked to symptoms of asthma, but are also considered as a risk factor for To compare with the situation in neighboring coun- tries we looked at the results of the world heath survey implemented in 2002–2003 by the WHO in 70 member states. Prevalence of suspected asthma BMC Public Health (2019) 19:663 Page 5 of 9 Table 2 Observed prevalence and estimation of the current prevalence of asthma, Reunion, 2016 Population observed % observed % estimated CI 95% Current asthma(a) 133 5.5 5.4 4.3–6.5 Overall 2419 100.0 100.0 (a)Over the last 12 months asthma: asthma diagnosed by a doctor, and/or a positive response in either of two questions “Have you ever been treated for asthma ”or "Have you been taking any medi- cations or treatment for asthma during the last 2 weeks and/or a positive response to “During the last 12 months have you experienced attacks of wheezing or whistling breath?”. The prevalence of asthma symptoms was 12.85 in Comoros, 12.40 in South Africa and 6.88 in Mauritius [14]. Although the definitions are not similar, it appears that the prevalence of asthma symptoms in Comoros and South Africa are close to the prevalence of sus- pected asthma in Reunion (12%) but nearly 2 times lower in Mauritius. Discussion The univariate analysis also revealed that other variables related to the presence of moisture and ambient temperature within the home, such as having obstructed air vents, a heater, or sources of moisture nearby were linked to asthma. The presence of cockroaches is also associated with a higher prevalence of asthma, and displays a dose- response relationship. This result is consistent with pre- vious studies [30–33]. We also found that suspected asthma shares some common risk factors with current asthma (such as obes- ity, having family members with asthma, passive smoke, the presence of mould or moisture in the home, and the presence of cockroaches). The prevalence of suspected asthma varied according to marital status and appeared Several environmental factors appear to be connected to the prevalence of suspected asthma (such as living close to a very busy road, a water treatment plant, or a source of harmful substances or air pollution). Discussion Individuals aged 18 to 45 years responded to questions related to asthma and related symptoms. This study did not use a definition of current asthma close to our but used the following definition for symptoms of Table 3 Risk factors for current asthma (multivariate), Reunion, 2016 Variables N Prevalence Ora(a) P-value 95% CI Gender Male 1080 3.5% 1.00 Female 1339 7.0% 1.97 3.62 10−4 [1.32; 2.96] Family member with asthnma No 1478 3.7% 1.00 Yes 917 8.1% 2.29 5.25 10−6 [1.53; 3.42] Length of time at curent residence Less than 5 years 1090 7.2% 3.51 1.13 10−3 [2.35; 5.25] 5 to 9 years 458 4.8% 2.35 2.58 10−3 [1.45; 3.79] 10 to 19 years 502 4.4% 2.04 2.80 10−2 [1.26; 3.31] 20 years or more 369 2.0% 1.00 Domestic heating No 2222 5.2% 1.00 Yes 197 8.1% 1.79 3.60 10−2 [1.04; 3.09] (a) Adjusted odds ratio Solet et al. BMC Public Health (2019) 19:663 Page 6 of 9 Table 4 Frequency of cockroach sightings in the home and prevalence of suspected asthma, Réunion, 2016 Total Population with suspected asthma % estimated suspected asthma 95% CI (p) OR 95% CI (OR) Throughout the year 428 55 14.1% [10.2%; 18.0%] 1.94 [1.21; 3.11] For 2 to 3 months a year 387 34 8.9% [5.6%; 12.2%] 1.16 [0.69; 1.96] A few times per year 1007 87 8.4% [6.4%; 10.4%] 1.09 [0.70; 1.70] Less than once a year 174 15 6.8% [2.4%; 11.2%] 0.87 [0.43; 1.75] Never 290 25 7.9% [4.3%; 11.5%] 1.00 [0.58; 1.73] Total 2286 216 9.3% [7,9%; 10,7%] to be higher among single, divorced, and widowed people. The result is in keeping with the results of the Health and Social Protection Survey (ESPS) conducted by IRDES in 2006 which found a prevalence of current asthma of 7.9% (6.8–9.0) among people living alone compared to 6.1% (5.3–6.9) among couples with no chil- dren, and 6.6% (6.1–7.1) among couples with children [14]. As with obesity, marital status is probably related to socioeconomic factors. In general, single-parent fam- ilies tend to have a lower standard of living and are more likely to suffer from poverty. asthma attacks and are suspected of affecting the long- term management of asthma [20–29]. (a)Odds ratio, adjusted Discussion One particularly interesting factor identified in this study concerns the use Table 5 Risk factors of suspected asthma (multivariate), Reunion, 2016 Criteria Answer N Prevalence Ora(a) P-value 95% CI Family member with asthma No 914 7.4% 1.00 Yes 1505 11.5% 1.48 2.22 10−3 [1.10; 2.27] Living close to a source of harmful substances or air pollution No 1625 6.9% 1.00 Yes 794 9.8% 1.33 3.33 10−2 [1.10; 2.08] Presence of smokers in the immediate social circle and mould in the home Presence of smokers in inner circle and mould in the home 458 14.0% 1.08 4.82 10−3 [0.82; 1.62] Presence of smokers in inner circle, but no mould in the home 798 7.5% 0.91 7.42 10−3 [0.75; 1.59] No smokers in inner circle, but mould in the home 387 7.8% 0.60 6.13 10−3 [0.37; 1.02] No smokers in the inner circle and no mould in the home 776 7.6% 1.00 Presence of mould in the home and used of insecticide sprays within the home Insecticide spray and mould in the home 268 10.8% 2.40 9.43 10−3 [1.42; 3.62] Insecticide spray, but no mould in the home 421 11.6% 2.02 7.51 10−3 [1.16; 3.54] No insecticide spray, but mould in the home 578 11.4% 2.99 7.73 10−3 [1.68; 4.31] No insecticide spray and no mould in the home 1152 6.2% 1.00 Marital Status Cohabitation 431 6.0% 1.00 Married or in a civil union 552 6.7% 1.60 1.81 10−2 [0.91; 2.59] Single 1366 10.5% 2.30 6.14 10−2 [1.37; 3.5] Divorced 52 10.6% 5.00 4.95 10−2 [1.39; 11.15] Widowed 18 15.0% 9.70 4.60 10−2 [1.25; 47.09] (a)Odds ratio, adjusted Table 5 Risk factors of suspected asthma (multivariate), Reunion, 2016 C i i A Solet et al. BMC Public Health (2019) 19:663 Solet et al. BMC Public Health (2019) 19:663 Page 7 of 9 Page 7 of 9 in Reunion with others published studies, and will be of value to health authorities and healthcare professionals. of insecticide sprays at home, with more frequent use be- ing associated with a higher prevalence of suspected asthma. Similar results can be found in the literature re- garding the link between the use of home aerosolised cleaning products and asthma or other respiratory symp- toms [34, 35]. This result is important in Reunion given the permanent risk posed by arboviruses outbreak and regular advice on preventing mosquitos’ bites [36, 37]. Conclusion The results of this first survey on the prevalence of asthma in the adult population of Reunion Island pro- vides novel information on the characteristics of asth- matic patients and on the environmental factors associated with diagnosed asthma and suspected asthma in this French territory. One of the major findings of this study is the propor- tion of individuals with suspected asthma who have not been medically diagnosed with asthma and who are not receiving any treatment. Consequently, we recommend the development of a strategy in conjunction with gen- eral practitioners to improve the diagnosis of asthma on Reunion, based on the support of general practitioners. Ongoing care and therapeutic education of diagnosed persons is also needed to reduce the burden of the disease. Our study has a number of limitations. The sampling methods excluded individuals with no landline or mobile phone, a group that makes up only a small portion (esti- mated at 3%) of the study population. The method used for selecting the interviewees partly consisted of repeat- ing the random call back process to generate the re- quired sample sizes for both categories of respondents (landline and only mobile); this may have introduced a selection bias insofar as the most available individuals were favoured for selection. The results are also consistent with the literature re- garding the links between asthma and environmental factors, such as the presence of moisture or cockroaches, and the use of insecticide sprays at home. These factors are important as exposure is common in tropical cli- mates such as that in Reunion. The study was conducted from the beginning of Feb- ruary to the end of May, outside the Austral winter (June to September) which may have led to an under- estimation of the prevalence as individuals might have been more inclined to report asthma symptoms during the colder months. However, the questionnaire asked about asthma attacks, treatments or symptoms experi- enced in the preceding 12 months, thereby including the Austral winter period. In terms of prevention, individual protection measures can be recommended, such as aerating and ventilating homes to reduce indoor moisture and mould growth, maintaining hygiene standards through cleaning, proper storage of foods to mitigate against insects or cock- roaches, and limiting the use of spray insecticides and household cleaning sprays at home. Discussion The result should be shared with health authorities in order to promote other methods of protection against in- sects (including mosquitoes) inside homes. Abbreviations d BMI: Body Mass Index; CATI: Computer Assisted Telephone Interview; CCEP: Internal expert committee of The French Public Health Agency for project evaluation; CI: Confidence Interval; CNIL: National Commission on Informatics and Freedoms; COPD: Chronic Obstructive Pulmonary Disease; ECRHS: European Community Respiratory Health Survey; EGEA: Epidemiological study on the Genetics and Environment of Asthma; ESPS: Health and Social Protection Survey; INSEE: French National Institute of Statistics and Economic Studies; IRDES: Institute for Research and Documentation in Health Economics; OR: Odd Ratio; WHO: World Health Organisation Furthermore, data about risk factors such as exposure to air pollution or allergens at home were not collected through objective measurements of air quality or allergy tests (such as skin prick tests or IgE blood tests), but ra- ther relied on answers to questions about environmental characteristics. Conclusion The prevalence estimates do not rely on a confirmed medical diagnosis including pulmonary function tests, but rather on self-reported symptoms or the prescription of medical treatment for asthma. For this reason, the classification is subject to possible reporting biases, espe- cially regarding exposure to risk factors, which depends on the participants’ status and ability to recollect past events. In order to reduce the reporting bias for asthma, all questions relating to symptoms were asked before mentioning asthma itself. Since the profile of those who refused to participate and the reasons for non- participation are unknown, it was impossible to compare respondents and non-respondents to assess the impact of any non-response bias. Metrological studies for air inside homes should be considered in cooperation with indoor environment medical advisers (a relatively new profession in Reunion) in order to improve our understanding of allergens present in domestic environments in tropical areas. Abbreviations BMI: Body Mass Index; CATI: Computer Assisted Telephone Interview; CCEP: Internal expert committee of The French Public Health Agency for project evaluation; CI: Confidence Interval; CNIL: National Commission on Informatics and Freedoms; COPD: Chronic Obstructive Pulmonary Disease; ECRHS: European Community Respiratory Health Survey; EGEA: Epidemiological study on the Genetics and Environment of Asthma; ESPS: Health and Social Protection Survey; INSEE: French National Institute of Statistics and Economic Studies; IRDES: Institute for Research and Documentation in Health Economics; OR: Odd Ratio; WHO: World Health Organisation Acknowledgments Despite these limitations, this epidemiological study using standardised questionnaires and definitions com- parable to those used in other national and international studies, enables us to compare the prevalence of asthma We would like to thank Drs Fabrice Paganin and Virgile Gazaille, hospital pulmonology practitioners at the University Hospital of Reunion, as well as Dr. Salem Atoui, liberal allergist and Dr. Marc Di Francia, liberal pulmonologist for their advice during the development of the survey questionnaire. Page 8 of 9 Page 8 of 9 Page 8 of 9 Page 8 of 9 Solet et al. BMC Public Health (2019) 19:663 Solet et al. BMC Public Health (2019) 19:663 Received: 29 October 2018 Accepted: 22 May 2019 Received: 29 October 2018 Accepted: 22 May 2019 Funding Th i 8. Delmas M-C, Guignon N, Leynaert B, Annesi-Maesano I, Com-Ruelle L, Gonzales L, et al. Prévalence et contrôle de l’asthme chez le jeune enfant en France. Rev Mal Respir. 2012;29:688–96. The entire study was funded by the Indian Ocean Health Agency. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. 9. Burney PG, Luczynska C, Chinn S, Jarvis D. The European Community respiratory health survey. Eur Respir J. 1994;7:954–60. Consent for publication Not applicable. 19. Trunk-Black Juel C, Suppli Ulrik C. Obesity and asthma: impact on severity, asthma control, and response to therapy. Respir Care. 2013;58:867–73. 20. Zock JP, Jarvis D, Luczynska C, Sunyyer J, Burney P. Housing characteristics, reported mold exposure, and asthma in the European community respiratory health survey (ECRHS). J Allergy Clin Immunol. 110:285–92. Competing interests The authors declare that they have no competing interests. 21. Sharpe RA, Bearman N, Thornton CR, Husk K, Osborne NJ. Indoor fungal diversity and asthma: a meta-analysis and systematic review of risk factors. J Allergy Clin Immunol. 2015;135:110–22. References 1. http://www.who.int/mediacentre/factsheets/fs307/en/. Date of access: April 10 2018. 2. Delmas M-C, Furhman C. L’asthme en France : synthèse des données épidémiologiques descriptives. Rev Mal Respir. 2010;27:151–9. 3. Delmas M-C, Guignon N, Leynaert B, Com-Ruelle L et al. Prévalence de l’asthme chez l’enfant en France. Archives de pédiatrie 2009. Vol. 16, n° 9 : 1261–1269. 4. Solet J-L, Catteau C, Nartz E, Ronat J, Delmas M-C. Epidémiologie de l’asthme à La Réunion : analyse de la mortalité (1990-1998) et de la morbidité hospitalière (1998-2002). Bull Epidemiol Hebd 2006. 2006;31:232– 4. 5. Fuhrman C, Nicolau J, Rey G, Solet J-L, Quenel P, Jougla E, et al. Asthme et BPCO : taux d’hospitalisation et de mortalité dans les départements d’outre- mer et en France métropolitaine, 2005-2007. Bull Epidemiol Hebd. 13(14): 168–72. 6. Prevot L, Dalleau-Passarelli N, Soulas A, Trevidic E. Prise en charge médicale des patients asthmatiques : enquête de pratique auprès d’assurés sociaux et de médecins de la Réunion. Rev Med Ass Maladie. 2003;34:3–13. 7. Martignon G, Catteau C, Debotte G, Duffaud B, Lebot F, Annesi-Maesano I. Allergies infantiles à l’île de La Réunion : existe-t-il des différences avec la métropole ? Rev Epidemiol Santé Publique. 2004;52:127–37. Availability of data and materials 10. Kauffmann F, Annesi-Maesano I, Liard R, Paty E, Faraldo B, Neukirch F, et al. Construction et validation d’un questionnaire en épidémiologie respiratoire. L’exemple du questionnaire de l’Etude Epidémiologique des facteurs Génétiques et Environnementaux de l’Asthme, l’hyperréactivité bronchique et l’atopie (EGEA). Rev Mal Respir. 2002;19:323–33. The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Authors’ contributions JLS wrote the study protocol, presented the project for approval to the internal expert committee (CCEP) of The French Public Health Agency, constituted and led the follow-up committee, monitored the implementa- tion of the project and drafted the manuscript. C R-S was a member of the follow-up committee of the study. As an expert in the field of asthma she made substantial contributions to the analysis and interpretation of the data collected. EM provided technical support to the development of the project: drafting the study protocol, sampling design, project monitoring. He was also a member of the follow-up committee. YLS was a member of the follow-up committee. As a statistician of The French Public Health Agency, he provided valuable advice on sampling and data analysis methods. AG was a member of the follow-up committee. She made substantial contributions to the conception and design of the survey. EB trained the investigators of the survey, implemented the sampling procedure and supervised the collection of the data. He also participated in the data analysis. NJ performed the multivariate analyses of the data and was a major contributor in revising the manuscript. LF was a member of the follow-up committee. He was involved in critically revising the manuscript and, as head of the Indian Ocean Region Regional Office of the French Public Health agency, supervised the work of the project manager and corresponding author. All authors read and approved the final manuscript. Ethics approval and consent to participate f f 11. Equipements en communications électroniques et audiovisuels des ménages et des individus La Réunion In: l’Autorité de Régulation de Communication Electronique et des Postes. Site disponible sur: https:// archives.arcep.fr/uploads/tx_gspublication/etude-equipements-usages-2012- Reunion-juil2013.pdf. At the time of the survey, the French law in force did not require the consultation of an ethics committee for this type of observational study that does not include any acts performed or products used on the participants. Until the decree n° 2016–1537 of November 16, 2016 modifying the law n° 2012–300 of March 5, 2012 relating to research on human subjects (Jardé law), this consultation was required only for biomedical research or research in routine care. 12. Sistek D, Tschopp JM, Schindler C, Brutsche M, Ackermann-Liebrich U, Perruchoud AP, et al. Clinical diagnosis of current asthma: predictive value of respiratory symptoms in the SAPALDIA study. Swiss Study on Air Pollution and Lung Diseases in Adults. Eur Respir J. 2001;17(2):214–9. In this context, the protocol of the survey, based on a data collection by phone directly from a random sample of individuals, was only approved by an internal expert committee (CCEP) of The French Public Health Agency. In addition, a simple declaration has been made to the National Commission on Informatics and Freedoms (CNIL) in accordance with the Data Protection Act, the only formality required by regulation. 13. www.irdes.fr/donnees/556-enquete-sur-la-sante-et-la-protection-sociale- 2012-etat-de-sante-1.xls. Date of access : November 10 2017. 14. To, et al. Global asthma prevalence in adults: findings from the cross- sectional world health survey. BMC Public Health. 2012;12:204. 15. Afrite A, Allonier C, Com-Ruelle L, Le Guen N. L’asthme en 2006 : prévalence, contrôle et déterminants. IRDES, vol. 117; 2011. prévalence, contrôle et déterminants. IRDES, vol. 117; 2011. Individuals contacted by phone to answer the questionnaire were informed of the optional nature of their participation and about their rights of access, rectification and opposition to the data concerning them. Informed consent was obtained from all participants. 16. Moffatt MF, Gut IG, Demenais F, Strachan DP, Bouzigon E, Heath S, et al. A large-scale, consortium-based Genomewide association study of asthma. N Engl J Med. 2010;363:1211–21. 17. Boulet L-P. Asthma and obesity. Clin Exp Allergy. 2013;43:8–21. 18. Sutherland ER. Linking obesity and asthma. Ann N Y Acad Sci. 2014;1311: 31–41. Author details 1S é bl 1Santé Publique France [The French Public Health Agency], Indian Ocean Regional Office, Saint-Denis, Reunion, France. 2Inserm U219, Institute of Public Health, Epidemiology, and Development (ISPED), Bordeaux University, Bordeaux, France. 3Agence de santé océan Indien [Indian Ocean Health Agency], Saint-Denis, Reunion, France. 4Santé publique France, [The French Public Health Agency], Saint-Maurice, France. 5Bureau d’études Synthèses [Synthesis analysis office], Saint-Denis, Réunion, France. 6Syntheses Mauritius LTD, Quatre Bornes, Mauritius. 22. Mendell MJ, Mirer AG, Cheung K, Tong M, Douwes J. Respiratory and allergic health effects of dampness, mold, and dampness-related agents: a review of the epidemiologic evidence. Environ Health Perspect. 2011;119: 748–56. 23. Kilpelainen M, Terho EO, Helenius H, Koskenvuo M. Home dampness, current allergic diseases, and respiratory infections among young adults. Thorax. 2001;56:462–7. Page 9 of 9 Solet et al. BMC Public Health (2019) 19:663 Solet et al. BMC Public Health (2019) 19:663 24. Evans J, Hyndman S, Stewart-Brown S, Smith D, Petersen S. An id i l i l d f h l i i f d h i i 24. Evans J, Hyndman S, Stewart-Brown S, Smith D, Petersen S. An epidemiological study of the relative importance of damp housing in relation to adult health. J Epidemiol Community Health. 2000;54:677–86. 24. Evans J, Hyndman S, Stewart-Brown S, Smith D, Petersen S. An y 25. Williamson IJ, Martin CJ, McGill G, Monie RDH, Fennerty AG. Damp housing and asthma: a case-control study. Thorax. 1997;52:229–34. 26. Pirhonen I, Nevalainen A, Husman T, Pekkanen J. Home dampness, moulds and their influence on respiratory infections and symptoms in adults in Finland. Eur Respir J. 1996;9:2618–22. Finland. Eur Respir J. 1996;9:2618–22. 27. Brunekreef B. Damp housing and adult respiratory symptoms. Allergy. 1992; 47:498–502. 27. Brunekreef B. Damp housing and adult respiratory symptoms. Allergy. 1992; 47:498–502. 28. Dales RE, Burnett R, Zwanenburg H. Adverse health effects among adults exposed to home dampness and molds. Am Rev Respir Dis. 1991;143:505–9. 28. Dales RE, Burnett R, Zwanenburg H. Adverse health effects among adults exposed to home dampness and molds. Am Rev Respir Dis. 1991;143:505–9. . Dales RE, Burnett R, Zwanenburg H. Adverse health effects among 29. Platt SD, Martin CJ, Hunt SM, Lewis CW. Damp housing, mould growth, and symptomatic health state. BMJ. 1989;298:1673–8. symptomatic health state. BMJ. 1989;298:1673–8. 30. Arruda LK, Vailes LD, Ferriani VP, Santos AB, Pomés A, Chapman MD. Cockroach allergens and asthma. J Allergy Clin Immunol. 2001 Mar;107(3): 419–28. 31. Arruda LK, Chapman MD. Author details 1S é bl The role of cockroach allergens in asthma. Curr Opin Pulm Med. 2001 Jan;7(1):14–9. 32. Sastre J, Ibanet MD, Lombardero M, Laso MT, Lehrer S. Allergy to cockroaches in patients with asthma and rhinitis in an urban area (Madrid). Allergy. 1996;51(8):582–6. 33. Fraser BN. Cockroaches in relation to bronchial asthma in the Durban area. S Afr Med J. 1979 Apr 14;55(16):637–8. 33. Fraser BN. Cockroaches in relation to bronchial asthma in the Durban area. S Afr Med J. 1979 Apr 14;55(16):637–8. 34. Le Moual N, Varraso R, Siroux V, Dumas O, Nadif R, Pin I, et al. Domestic use of cleaning sprays and asthma activity in females. Eur Respir J. 2012;40: 1381–9. 35. Bédard A, Varraso R, Sanchez M, Clavel-Chapelon F, Zock J-P, Kauffmann F, et al. Cleaning sprays, household help and asthma among elderly women. Respir Med. 2014;108:171–80. 36. D’Ortenzio E, Balleydier E, Baville M, Filleul L, Renault P. Dengue fever in the Reunion Island and in South Western islands of the Indian Ocean. Med Mal Infect. 2011. 37. Renault P, Solet JL, Sissoko D, Balleydier E, Larrieu S. Filleul, et al. a major epidemic of chikungunya virus infection on Reunion Island, France, 2005– 2006. Am J Trop Med Hyg. 2007;77:727–31. 37. Renault P, Solet JL, Sissoko D, Balleydier E, Larrieu S. 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https://openalex.org/W2080931451	https://zenodo.org/records/2479966/files/article.pdf	German	null	Über das Verhalten permanenter Magnete bei Änderung des äußeren magnetischen Widerstandes	Annalen der Physik	1,909	public-domain	5,480	1) H. Eichel, Dissertation Halle 1908. 553 553 y p 3) E. Rempken, Dissertation Tubingen 1906 und Ann. d. Phys. 20. p. 1017. 1906. , 2) R. H. Weber, Ann. d. Phys. 16, p. 172. 1905. p Annalen der Phiaik. IV. Folge. 28. 37 1) H. Eichel, Dissertation Halle 1908. 2) R. H. Weber, Ann. d. Phys. 16, p. 172. 1905. 3) E. Rempken, Dissertation Tubingen 1906 und Ann. d. Phys. 20. p. 1017. 1906. Annalen der Phiaik. IV. Folge. 28. 37 6. Uber das 'Perhaltem permamenter Magmete be% Anderzcmg des auperem magmetischen W%derstandee; vom Paul Gehme. (Auszug aus der Dissertation des Verfassers, Halle 1908.) (Auszug aus der Dissertation des Verfassers, Halle 1908.) Vorliegende Untersuchungen beschaftigen sich zunachst mit der Frage: Was bleibt in einem permanent magnetischen Kreise bei Anderung des (au5eren) magnetischen Widerstandes konstant? Die im physikalischen Institut der Universitat Halle angestellten Untersuchungen H. Eich els hatten eine Konstanz der Kraftlinienanzahl ergeben, wahrend R. H. Web e r und spater E. K e mpken 3, ebenfalls auf Grund experimenteller Untersuchungen die magnetomotorische Kraft konstant , die Kraftlinienzahl dagegen variabel finden. Zur Untersuchung gelangten durchweg kreisformig an- geordnete Systeme aus Stahl und Eisenstucken verschiedener Kombination und GroBe. Als Material zum Aufbau der Systeme, fur die ich die Versuchsergebnisse im folgenden mit- teilen will, dienten eine Anzahl stahlerner Ringe. 1. Ein Ring mit kreisformigem Querschnitt aus Wolframstahl von der Firma Gebr. Bohler & Co., Wien (Marke ME): Achsendurchmesser . . . . . 14,032 cm Querschnittsdurchmeeser. . . . 1,3318,, Querschnitt . . . . . . . . 1,3931,, Dieser Ring wurde nach dem Harten in zwei Halbringe zer- schnitten. 37 P. Gehne. 554 2. Zwei Halbringe aus Remystahl von bedeutend groberen Dimensionen, aber gleichfalls mit kreisformigem Querschnitt : Achsendurchmesser . . . . . . . 17 ern Querschnittsdurchmesser . . . . . 5 ,, 3. Ein Ring mit quadratischem Querschnitt aus Wolfram- stahl von Bohler & Co.: 3. Ein Ring mit quadratischem Querschnitt aus Wolfram- stahl von Bohler & Co.: Achsendurchmesser . . . . . 14,0878 cm Qucrschnitt . . . . . . . . 1,834 qcm Dieser Ring wurde zunachst im unzerschnittenen Zustande untersucht und dann ebenfalls in zwei Halbringe zerschnitten. Nach dem Zerschneiden wurden in jedem Falle die Schnitt- 0lchen sorgfaltig eben geschliffen, urn einen guten magne- tischen SchluE zu ermoglichen und dann die Ringe magne- tisiert. Fur die unter 1. aufgefuhrten Halbringe wurde zu ihrer Montierung ein Schlittenapparat aus Messing hergestellt, der es ermoglichte, den Abstand der beiden Halbringe mittels einer Mikrometerschraube zu regulieren und zu messen. Die Halb. ringe waren so montiert, dab man eine Probespule sukzessiv liber den ganzen Umfang des magnetischen Kreises fuhren konnte, ohne da6 die einzelnen Magnetstucke ihre gegenseitige Lage anderten. Der unter 3. aufgefiihrte gr6Eere Ring befand sich auf einem einfacheren Schlittenapparate aus Holz und war so be- festigt, daE nur je eine Hllfte der beiden Halbringe der Unter- suchung zuganglich war. Hier erfolgte die Bestimmung der Schlitzbreiten durch Glasskalen. Als Probespulen dienten zwei verschiedene Arten von Spulen. 1. 1) Korrektionen berechnet nach Kohlrausch, Lehrbuch d. prakt. Phyeik p. 472. Leipzig 1905. 37 * 6. Uber das 'Perhaltem permamenter Magmete be% Anderzcmg des auperem magmetischen W%derstandee; vom Paul Gehme. (Auszug aus der Dissertation des Verfassers, Halle 1908.) Spulen mit nur wenigen Windungen, und zwar fur jeden Ring besondere. Sie hatten einen Querschnitt, der nur wenig grober war als der des betreffenden Ringes, so daB man sie auf diesem entlang hin und her verschieben konnte. 1. Spulen mit nur wenigen Windungen, und zwar fur jeden Ring besondere. Sie hatten einen Querschnitt, der nur wenig grober war als der des betreffenden Ringes, so daB man sie auf diesem entlang hin und her verschieben konnte. 2. Einige mit besonderer Sorgfalt aus zahlreichen Win- dungen eines dilnnen Drahtes hergestellte kleine Probespulen, deren Windungs0Bche ich mit moglichster Genauigkeit auf 555 Perhalten permanenter Magnete usw. geometrischem und elektromagnetischem Wege bestimmte. Die naheren Angaben iiber sie enthalt die folgende Tabelle. geometrischem und elektromagnetischem Wege bestimmte. Die naheren Angaben iiber sie enthalt die folgende Tabelle. Tabelle 1. Tabelle 1. Windungsfllchen der benutzten Probespulen. Windungsfllchen der benutzten Probespulen. Spde 1 ,, 11 ,, I11 0,3514 0,4829 0,6684 0,7355 0,9500 1,0786 I I I I 0,1690 0,1835 0,1853 I , l a l f l geom. clektrom. ) I f Mittel 4,845 19,84 19,85 19,85 7,03 58,53 58,20 58,36 8,89 113,76 114,2 114,O Genaueres uber Herstellung dieser Spulen, sowie Qer- suchsanordnung und Konstruktion der verwendeten Apparate findet sich in meiner Dissertation. Als MeBinstrument diente ein Deprez d ’ k s o n v a l - Galvanometer von Edelmann, zur Eichung ein Erdinduktor. Zur Untersuchung wurden nebeneinander zwei Methoden angewendet. Die erste Methode entspricht im wesentlichen der von Eichel benutzten. Eine mit dem Galvanometer verbundene Probespule der ersten Art wird vom indifferenten Punkte des einen Halbringes sukzessive bis zur Mitte des einen Spaltes gefiihrt. Durch Addition der auf absolutes MaB reduzierten Galvanometer- ausschlage, die bei einer jeden Verschiebung erfolgen, erhalte ich die Anzahl der aus der Peripherie des Magnethalbringes austretenden Kraftlinien. Aus den untereinander nur wenig abweichenden Werten der linken und rechten Halfte eines Halbringes wurde das Mittel genommen. In der nachstehenden Tabelle sind die auf diese Weise gefundenen Werte fur den einen Halbring unter Kolumne ,I, far den anderen Halbring unter Kolumne I1 angegeben. Durch Herausziehen der Spule aus einem der beiden Spalte mi& man die im Ferromngne- tikum verlaufenden Kraftlinien ; die entsprechenden Werte 37 656 P. Gehne. finden sich in der Tabelle unter ,,Spalt". Durch Addition dieser Werte zu I bzw. I1 erhalt man die Anzahl der gesamtcn Kraftlinien fur Halbring I und 11. Diese Werte sind in der Tabelle als 21 bzw. 211 angefiihrt. finden sich in der Tabelle unter ,,Spalt". Durch Addition dieser Werte zu I bzw. I1 erhalt man die Anzahl der gesamtcn Kraftlinien fur Halbring I und 11. Diese Werte sind in der Tabelle als 21 bzw. 211 angefiihrt. Diese Untersuchungen wurden zunachst an den unter 1. aufgefiihrten kleineren Halbringen vorgenommen, und zwar bei verschiedenen Spaltbreiten. Dann untersuchte ich noch jeden Halbring fiir sich einzeln durch plotzliches Abziehen der Probespule vom indifferenten Punkte aus; diese Werte finden sich unter Spalt = 00. Die folgende Tabelle enthalt die Resultate einer solchen Versuchsreihe, die Werte der Kraftlinienzahlen sind in ab- solutem Ma6e (Maxwell) angegeben. Tabelle 2. Variation der Kraftlinieneahlen fur Ringeyetern 1. 1) Dissertation des Verfassers p. 35 ff. Variation der Kraftlinieneahlen fur Ringeyetern 1. 195 3,0 495 690 895 m 4726 5546 6036 6336 6579 7134 I1 4655 5472 5955 6243 6495 7080 2994 2112 1606 1281 983 0 7720 7658 7642 7617 7562 7134 7649 7584 7561 7524 7478 7080 Wir bemerken also eine deutliche, wenn auch relativ geringe Abnahme der Kraftlinienanzahl bei VergroSerung des magnetischen Widerstandes. Qanz bedeutend auffglliger tritt dies Verhalten bei Unter- suchung der unter 2. angegebenen groBeren Halbringe in Er- scheinung. Versuchsanordnung und Ausfiihrung der Versuche ist analog dem oben Dargelegten. Die folgende Tabelle ent- htilt die Resultate einer Versuchsreihe mit diesem groBeren System; hier sind die Resultate in nicht reduzierten Galvano- meterausschlagen angegeben. Der Wert des Reduktionsfaktors ist 40,91. Perhalten permanenter Maynete usw. Perhalten permanenter Maynete usw. 557 p y 3 6 9 12 15 1s 21 21 27 30 200,Q 249,2 265,7 276,9 284,O 289,6 293,O 293,4 294,l 294,9 Tabelle 3. Variation der Kraftlinienzahl fur Ringsystem 2. Spaltbreite in mm I1 181,s 229,l 245,6 256,5 263,7 269,O 27 4 6 271,7 272,O 272,5 273,7 269,3 Spalt 415,2 284,2 229,9 177,l 148,6 124,5 107,6 94,s S4,2 74,9 56,l 0 21 616,l 495,6 454,O 432,6 414,l 400,6 388,2 369,s 352,2 533,4 378,3 29a,o 2 IT 596,O 513,3 475,5 433,6 412,3 393,5 378,6 366,5 356,2 347,4 329,s 269,3 Tabelle 3. Variation der Kraftlinienzahl fur Ringsystem 2. Weitere Versuche ergaben, da0 die relative hderung der Kraftlinienzahl von der StarRe der permaiienten Maynetisierung so gut wie unabhiingig ist.k) Demnach diirfte die Meinung Kempkens, die abweichenden Resultate Eichels hingen mit der hohen Sattigung der von ihm benutzten Magnete ab, wohl nicht stichhaltig sein. Meiner Meinung nach e r k k e n sich die abweichenden Resultate aus folgender Uberlegung. J e kleiner der Qnerschnitt der Magnete im Verhaltnis zu ihrem Achsen- durchmesser ist, desto groBer ist die relative Verbreiterung des Querschnittes des Kraftlinienbiindels bei SpaltvergrOBerung. Schon dae verschiedene Verhalten des groBen und kleinen Ringsystems scheint darauf hinzudeuten und eine Reihe von Versuchen iiber Streuung und die dadurch bewirkte Wider- atandsverminderung eines Luftspaltes, die ich anstellte, machen diese Annahme noch wahrscheinlicher. Die von Eichel benutzten Magnete haben noch vie1 kleinere Querschnitte (0,31-0,41 qcm) als die vou mir be- nutzten kleinsten (1,36-1,39 qcm). Die geringen Lderungen der Spaltbreite, die Eichel vorgenommen hat, wurden durch 558 P. Gehne. die Querschnittsverbreiterung so weit kompensiert, daB eine Anderung der Kraftlinienzahl innerhalb der Beobachtungs- genauigkeit nicht mehr zu konstatieren war. 1) E. Kempken, Tubinger Dissertation p. 23. 1906 und Ann. d. Phys. 20. p. 1028. 1906. Variation der Kraftlinieneahlen fur Ringeyetern 1. g g Einen anderen von Kempkenl) angegebenen Versuch wiederholte ich mit wesentlich anderem Erfolge. Eine Spule aus vier Windungen von 17 cm Durchmesser wurde im Spalt aufgestellt und plStzlich herausgezogen. (Kempken benutzt eine solche von nur einer Windung und 11 cm Durchmesser bei gleicher Dimensionierung des Magnetsystems.) Bei einer Spaltbreite von 1,l cm erfolgte ein Ausschlag von 493,3 Skt., bei einer Spaltbreite von 4,0 cm erfolgte ein solcher von 271,O Skt. Differenz = 222,3. Differenz = 222,3. Auf der indifferenten Zone des Halbringes 1 waren eben- falls vier Windungen festgelegt. Plotzliche Spaltverkleinerung von 4,O cm auf 1,l cm ergab einen Ausschlag von 146,2 Skt. Bei dem von Kempken angestellten Versuche stimmte dieser Wert mit obiger Differenz uberein. mit obiger Differenz uberein. Es sollte damit bewiesen werden, daS tatsachlich die l der Kraftlinien nicht konstant sei, und daB die Ver- '. 1 bderung der Feldstarke im Spalt nicht durch mehr oder weniger groBe Streuung zu erkliiren sei, wie Eichel be- hauptet hatte. Tatsachlich umfaBt die im Spalt auf- gestellte groEe Spule wenig- stens fur mein Magnetsystem nicht samtliche Kraftlinien, wieKempken annimmt son- Es sollte damit bewiesen werden, daS tatsachlich die Anzahl der Kraftlinien nicht konstant sei, und daB die Ver- '. 1 bderung der Feldstarke im Spalt nicht durch mehr oder weniger groBe Streuung zu erkliiren sei, wie Eichel be- hauptet hatte. Tatsachlich umfaBt die im Spalt auf- gestellte groEe Spule wenig- stens fur mein Magnetsystem nicht samtliche Kraftlinien, wieKempken annimmt, son- dern, wie aus Fig. 1 ersicht- lich ist, nur die Kraftlinien a und b , nicht aber c. Fur die von Kempken benutzte Spule, die einen geringeren Quer- schnitt hat, diirfte seine Annahme noch weniger zutreffen. Eine durchgehende Ubereinstimmung obiger Werte kijnnte nur Fig. 1. Es sollte damit bewiesen werden, daS tatsachlich die Anzahl der Kraftlinien nicht konstant sei, und daB die Ver- '. 1 bderung der Feldstarke im Spalt nicht durch mehr oder weniger groBe Streuung zu erkliiren sei, wie Eichel be- hauptet hatte. Tatsachlich umfaBt die im Spalt auf- gestellte groEe Spule wenig- Fig. 1. Fig. 1. Verhalten permanenter Magnete usw. 559 dann eintreten, wenn die in Fig. 1 mit c bezeichneten Kraft- linien entweder iiberhaupt nicht vorhanden waren oder wenig- stens ihre Anzahl sich nicht anderte, d. h. keine Kraftlinien von der Lage b in die Lage c iibergingen. Die bisher benutzten Magnetstucke unterwarf ich dann noch einer anderen, zuerst von R. H. Weberl) angegebenen und spater auch von E. Kempken2) angewandten Untersuchungs- methode. Diese Methode besteht darin, daS man eine Probe- spule, deren Querschnitt kleiner ist als der der benutzten Toroide, in einem der MeBspalte zentrisch anbringt. La6t man diese Spule pliitzlich aus dem Spalt herausfallen, so ist der hierdurch hervorgerufene Ausschlag des ballistischen Gal- vanometers proportional der Anzahl d a geschnittenen Kraft- linien also proportional dem Kraftlinienstrom in der durch die Dimensionen der Spule bestimmten Induktionsrohre. 3) It. Gans, Einfuhrung in die Theorie des Magnetismus p.63. p Leipzig 1908. 1) R. H. Weber, Ann. d. Phys. 16. p. 178. 1905. 2) E. Kempken, Tiibinger Dissertation nnd Ann. d. Phys. 20. p. 1017. 1906. 1) R. H. Weber, Ann. d. Phys. 16. p. 178. 1905. 2) E. Kempken, Tiibinger Dissertation nnd Ann. d. Phys. 20. 3) It. Gans, Einfuhrung in die Theorie des Magnetismus p.63. p. 1017. 1906. Leipzig 1908. Differenz = 222,3. Diese Messungen werden nun fur eine grbBere Anzahl von Spalt- breiten durchgefiihrt. Mi6t man auBerdem fur jeden Versuch die Liinge der zugehorigen Luftspalte, so hat man andererseits Proportionalwerte fur die Widerst‘ande, die sich zum inneren Widerstande wi des Ferromagnetikums hinzuaddieren ; denn solange gewisse Grenzen nicht iiberschritten werden, kann der Widerstand eines solchen Luftspaltes seiner Lange proportional gesetzt werden. Fur das Feld im Schlitz Qa gilt folgende Qleichung: 16 4nmnz,- 14 - 4- I, Pr k . 8) Qa = Unter der Annahme eines konstanten inneren Widerstandes und unter Annahme einer konstanten magnetoniotorischen Kraft mussen sich die nach obiger Methode erhaltenen Galvano- meterausschlage darstellen lassen nach der Formel y p 2) E. Kempken, Tiibinger Dissertation nnd Ann. d. Phys. 20. p. 1017. 1906. 2) E. Kempken, Tiibinger Dissertation nnd Ann. d. Phys. 20. p. 1017. 1906. 3) It. Gans, Einfuhrung in die Theorie des Magnetismus p.63. p Leipzig 1908. 3) It. Gans, Einfuhrung in die Theorie des Magnetismus p.63. p Leipzig 1908. 560 P. Gehne. nachdem man aus zwei Beobachtungen die beiden unbekannten Konstanten a und C berechnet hat. In obiger Formel ist a eine GroBe, die dem inneren Widerstande des Ferromagnetikums entspricht. Bezeichnet man mit 1 die Gesamtlange der vorhandenen Luftspalte in Millimetern, so mu8 a die dem inneren Widerstande des Ferro- magnetikums entsprechende Luftstrecke ebenfalls in Millimetern angeben, wenn man annimmt, daB der Querschnitt der be- trachteten zentralen Induktionsrohre uberall der gleiche ist. 1st li die Lange der Induktionsrohre im Ferromagnetikum in Millimetern, so muB sein: li - a also pT = -. P. a 4 _ - li - a also pT = -. P. a 4 _ - Die Resultate einer Versuchsreihe und zwar fiir das gro6e Magnetsystem seien in folgender Tabelle wiedergegeben. Die Spaltbreite, bei der die Beobachtung 1 stattfand, war mit Stangenzirkel und Glasskala genau festgestellt. Sie betrug : links 0,34 cm rechts 0,39 cm. links 0,34 cm rechts 0,39 cm. links 0,34 cm rechts 0,39 cm. Das entspricht einer Gesamtluftstrecke von 0,73 cm. Fur jede weitere Beobachtung verschob ich den beweglichen Halbring um 0,2 cm, Die erhaltenen Ausschlage Q! sind in Skalenteilen angegeben. In der folgenden Tabelle bedeutet S die Ver- schiebung des beweglichen Halbringes in Millimetern yon der Anfangsstellung aus. g g Als Probespule diente Spule I1 p. 555. p p p Die Konstanten a und I sind aus den Beobachtungen 2 und 6 berechnet. 1) Diasertation des Verfassers p. 18. Differenz = 222,3. Also C a+ 11,3 ’ 388,s = ~ C a + 27,3 ’ 209,6 = - wo fur I die gesamte Lange der Luftspalte in Rechnung ge- setzt ist. Es ergibt sich: wo fur I die gesamte Lange der Luftspalte in Rechnung ge- setzt ist. Es ergibt sich: C = 7267,3 a = 7,42. Perhalten permanenter Magnete usw. 561 p g Tabelle 4. Konstanz der magnetomotorischen Kraft fur Ringsystem 2. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 - 8 in mm 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 OL beobachtet 492,s 388,8 319,6 272,2 237,O 209,6 187,4 169,l 156,l 143,O 132,s 124,O 116,2 104,4 110,o 99,7 94,7 90,4 87,l 83,6 80,5 a berechnet 494,6 388,s 320,4 272,4 236,9 209,6 170,4 155,s 143,5 133,O 124,O 116,O 102,9 92,5 88,O 83,9 80,2. 76,s 188,O 109,l 97,4 Fehler + 1,7 090 +0,8 + 0,2 -0,l 0,O 4- 0,6 + 1,3 4- 0,5 - 0,3 + 0,l 070 - 0,2 -0,9 - 1,5 -2,3 - 2,2 -2,4 - 3,2 - 3,4 - 3,7 Fehler in Proz. + 0,s 0 8 +0,3 +0,1 0,0 0,0 + 0,3 + 0,8 -0,2 + 0,3 + 0,l 070 - 0,2 - 0,s - 1,5 - 2,3 - 2,4 - 2,7 - 3,s - 4,3 - 4.8 Tabelle 4. Bis zur Beobachtung 13 ist die Ubereinstimmung yon a berechnet und a beobachtet gut, yon da ab beginnt eine systematische Abweichung. Die Ergebnisse stimmen im wesentlichen mit denen E. Kempkens uberein, auch E. Kempken vermutet schon, daB die Abweichungen durch eine Verbreiterung der zentralen Induktionsrohre nach der Mitte des Spaltes zu und durch die dadurch bedingte Widerstandsverminderung des Luftspaltes zu erkliren sein. DaB diese Abweichungen bei den mit den kleineren Ringsystemen angestellten Versuchen schon bei ge- ringeren Spaltbreiten eintreten und zwar um so fruher, je gro6er der Querschnitt der benutzten Probespule war, machte diese Annahme noch wahrscheinlicher. l) 562 P. Gehne. P. Gehne. SchlieBlich ging ich dazu uber, diese durch Verbreiterung der Induktionsrohre herbeigefuhrte Widerstandsverminderung zu bestimmen. Ich stellte diese Versuche fur drei bestimmte Spaltbreiten an, die den Beobachtungen 15, 17 und 21 obiger Tabelle entsprechen. Ich benutzte zu diesem Zweck jedesmal die drei auf p. 555 beschriebenen Induktionsspulen und brachte jede einzelne nacheinander an funf verschiedene Stellen des Luftspaltes, und zwar so, daI3 die Mittelpunkte ihrer Windungs- flachen sich auf der Achse der zentralen Induktionsrohre be- fanden. Auf diese Weise bestimmte ich das Feld an den ver- schiedenen Stellen des Spaltes und konnte so feststellen, wie vie1 Kraftlinien an jeder Stelle aus der zentralen Induktions- rohre ausgetreten waren und uber welchen Bereich sie sich ausgedehnt hatten. g Auf diese Art kann man die Gestalt und demnach den Widerstand der verbreiterten Induktionsrohre graphisch und rechnerisch bestimmen. Fiihrt man die so reduzierten Wider- standswerte in die Formel a! = Cl(a + I ) ein, so ergibt sich (vgl. Tab. 4) fur Beobschtung 15 . . . a berechnet 104,3 a beobachtet 104,4 Fehler -0,l Proz. 9 ) ,I 17 . . . a berechnet 94,4 11 ,? 20 . . . a berechnet 80,l a beobachtet 94,7 ,, -0,3 ,, a beobachtet 80,5 ,, -0,5 ,, Wir erhalten also wieder eine genugende Ubereinstimmung von a berechnet mit a beobachtet und damit einen Nachweis der Konstanz der magnetomotorischen Kraft uber den Bereich der bisherigen Beobachtungen hinaus. g g Zum SchluB sei noch eine Reihe von Versuchen erwahnt, die ich mit dem unter 3. p. 554 angefuhrten Rihge anstellte. Der Ring wurde zunachst mit einer Sekundarwickelung aus 692 Windungen eines 0,5 mm dicken isolierten Drahtes in einer Lage und daruber mit einer Primarwickelung aus 623 Windungen eines 1 mm starken Drahtes in drei Lagen versehen. Mit Hilfe der ballistischen Methode wurde so in bekannter Weise eine vollstandige Hysteresiskurve aufgenommen. V h lt t M t 563 Verhalten permanenter Magnete usw. 563 Verhalten permanenter Magnete usw. 563 Nachstehende Tabelle enthalt die Ergebnisse fur den auf- steigenden Ast. Tabelle 5. Magnetisierungekurve fur Ring 3 (unzerschnitten). -- Magnetisierungekurve fur Ring 3 (unzerschnitten). 77 75 79 84 94 94 106 105 114 117 123 i in Amp. P. Gehne. 0,18 0,50 0373 0,86 1,04 1,33 1,50 1,69 2,06 1,90 2,21 2,40 42,45 5453 128 2,87 50,77 7202 142 3,19 56,43 8289 147 3,60 63,68 9476 149 4,12 72,88 10686 145 4,80 84,91 11746 138 5,81 102,8 12802 125 7,30 129,l 13789 109 9,70 171,6 14542 87 14,50 256,5 16107 63 8 3,184 8,844 12,91 15,21 18,40 23,53 26,53 29,89 33,61 36,44 39,09 -- B 246,3 662,O 1020 1273 1727 2219 2812 3151 3841 4270 4801 Nach erfolgter Entmagnetisierung wurde der Ring zer- schnitten und von neuem bewickelt. Die Sekundarwickelung bestand aus 652 Windungen in einer Lage; die Primarwickelung aus 607 Windungen in drei Lagen. Zwischen die beiden Ringhalften waren kleine Hartgummi- scheibchen von gleichem Querschnitt wie der Ring eingefiigt. Auf diese wurden je 20 Windungen eines 0,13mm dicken iso- lierten Drahtes aufgewickelt. Seitlich angeklebte Glimmer- blattchen verhuteten ein Abgleiten der Windungen. Diese beiden Spulen wurden hintereinander geschaltet. Durch sie sollte die Induktion auch im Interferrikum bestimmt werden. Der Spalt zwischen den Polfliichen betrug 1,6mm. Bei der Berechnung von $j sowohl wie von b, wurden die gleichen Formeln angewendet, die fur den vollstandigen magnetischen Kreis gultig sind. Es sollte durch einen Ver- gleich der so gewonnenen Resultate mit denen far den un- zerschnittenen Ring ein Anhalt dafiir gefunden werden , wie gro6 die durch die Vernnchlassigung entstandenen Fehler ins- besondere fur den Wert $= 23JQ sind. Die folgende Tabelle gibt wieder die Resultate fur den aufsteigenden Ast der Kurve. !B2 ist die Induktion im Interferrikum. 564 P. Gehne. i in Amp. O,47 O,60 1,73 2,53 3,29 4,OO 4,69 5,98 7,16 8,22 10,13 12,83 14,40 5,35 Tabelle 6. Magnetisierungskurve fur Ring 3 (zerschnitten). 8,1 10,3 29,8 43,6 56,7 68,9 YO,8 92,2 103,l 123,4 141,7 174,6 248,2 221,l % 243,4 338,O 1287,O 2185,O 3173,O 4169,O 5106,O 5918,O 6583,O 7509,O 8087,O 8778,O 9408,O 9678,O 1132 203,8 277,O 671,8 1188,O 1745,O 2477,O 3213,O 3897,O 4901,O 5668,O 6217,O 6960,O 7739,O 8109,O P' 30 33 43 50 56 60 63 64 64 G 1 57 50 43 39 1,19 1,22 1,92 l,S4 1,68 1,59 1,52 152 1,34 1,53 1,30 1,?6 1,22 1,19 Tabelle 6. Magnetisierungskurve fur Ring 3 (zerschnitten). Magnetisierungskurve fur Ring 3 (zerschnitten). Die Resultate der magnetischen Kreisprozesse und ebenso das Verhalten von p und p' ist auBerdem noch kurvenmaSig dargestellt worden (Fig. 2 und 3). Ein Vergleich der beiden Kurven fiir p und p' gibt AufschluB uber die durch Vernach- lassigung entstandenen Fehler. 1) R. Gans, Ann. d. Phys. 22. p, 481. 1907; Elektrotechn. Zeitschr. 2) R. Gans, Einfuhrung in die Theorie des Magnetismus. Leipzig 28. p. 527. 1907. 1908. P. Gehne. Diese sind relativ groB, p' ist teilweise kleiner als die Halfte von p. Abweichend von dieser bisher besonders in der Technik ublichen GroBe p hat R. Gansl) in einer Reihe yon Arbeiten, die in letzter Zeit erschienen sind, eine andere experimentelle Konstartte eingefiihrt, die er spaterhinz) mit dem Namen reversible Permeabilitat (p,,) benennt. Diese fur permanente Magnete gultige GroSe wird durch Anwendung sehr schwacher Zusatzfelder bestimmt und ergibt sich fur permanente Magneti- sierungen verschiedener Starke als ziemlich konstant. Urn diese GroBe pT auch fur den zuletzt besprochenen Ring zu bestimmen, wurde er mit 652 Windungen sekundiirer und 604 Windungen primarer Wickelung versehen. Als diese Perhalten permanenter Magnete usw. 565 Bestimmung durchgefuhrt werden sollte, war der Ring bereits zerschnitten, beide Halften wurden mit ihren Polflachen fest an- einander gepreSt , im ubrigen wurden dieselben Vernachlassigungen be- gangen wie bei der Be- stimmung von b, bzw. p'. Den Primarstrom wahlte ich so klein, daB das durch ihn erzeugte Feld keinerlei Remanenz hervorrief, er betrug 0,00279 Amp. Die durch Kommutierung dieses Stxo- mes erzeugte Feldande- rung betragt A@=O,O9570. Dem entsprach eins An- derung d B = 2,777. Es folgt demnach Fig. 2. Fig. 2. hrt er n, mit n- m en e- e- p'. m aB gte nz ug ch xo- e- 70. n- Es ng bis Fig. 2. 1W 1IO 120 130 1W 150 160 110 180 190 200 ?lo 220 230 240 L90 260 210 280 Fig. 3. ereits zerschnitten, Halften wurden mit Polflachen fest an- er gepreSt , im n wurden dieselben hlassigungen be- wie bei der Be- ung von b, bzw. p'. en Primarstrom ich so klein, daB urch ihn erzeugte keinerlei Remanenz rief, er betrug 9 Amp. Die durch utierung dieses Stxo- erzeugte Feldande- etragt A@=O,O9570. entsprach eins An- d B = 2,777. Es emnach diese Beobachtung r Magnet vorher bis Fig. 2. 80 90 1W 1IO 120 130 1W 150 160 110 180 190 200 ?lo 220 230 240 L90 260 210 280 Fig. 3. Fur diese Beobachtung war der Magnet vorher bis Fig. 2. 80 90 1W 1IO 120 130 1W 150 160 110 180 190 200 ?lo 220 230 240 L90 260 210 280 Fig. 3. Fig. 3. P. Gehne. 566 zur Sattigung magnetieiert gewesen. Bei einer geringeren Magnetisierung ergab sich p,. = 29,2 und nach vollstandiger Entmagnetisierung p,. = 29,O. Also im Mittel pr = 29,l. p,. = 58. p,. = 58. Fur diese beiden in obigen charakterisierten Magnethalb- ringe stellte ich nun eine Reihe von Versuc.hen nach der auf p. 559 angegebenen Methode an. Bei dem geringen Quer- schnitt der Magnete muBte die zur Verwendung kommende Probespule mSglichst klein dimensioniert aein. Sie bestand aus 80 Windungen eines 0,l mm atarken isolierten Drahtes. Ihre Dimensionen waren folgende: r0 = 1,195 mm ; r1 = 3,75 mm . r0 = 1,195 mm ; r1 = 3,75 mm . P. Gehne. Nimmt man nun an, daS die dnrch Vernachlassigung (Streuung und entmagnetisierende Kraft) entstandenen Fehler etwa die gleichen sind als die bei der Berechnung von p’ unter analogen Verhaltnissen untergelaufenen, also im ungiinstigsten Falle sich etwa auf 100 Proz. belaufen, so kommen wir zu der korrigierten Gro8e Daraus f = 16,73 cm2. Fur die erste Versuchsreihe wurden die mit einer Lage aus 229 Windungen versehenen Magnetstucke mit einem Strom von 5 Amp. magnetisiert , bei der zweiten Versuchsreihe mit 15 Amp., bei der dritten mit 25 Amp. Durch den letzteren war die praktische Sattigung erreicht, denn durch Strome bis zu 40 Amp. war eine Steigerung der Magnetisierung nicht mehr zu erzielen. Die drei folgenden Tabellen geben die Ergebnisse der drei angestellten Versuchsreihen. Die Art der Berechnung ist die gleiche wie friiher (p. 559ff.). In Tabb. 8 und 9 sind die Konstanten a und C aus den Beobachtungen 1 und 4 aus- gerechnet. Fur Tab. 7 ist eine Berechnung der Werte von oc nicht durchgefuhrt. Es bietet sich uns das gleiche Bild wie fruher. Anfanglich haben wir gute Ubereinstimmung der Werte a berechnet und cz beobachtet, spaterhin die durch Ver- breiterung der Induktionsrohre verursachte Abweichung. Die Werte sind in Skalenteilen angegeben. 567 Perhalten permanenter Magnete usw. p g Tabelle 7. Magnetisiert mit 5 Amp. 1 2 3 4 5 6 1 8 9 1,15 2,25 2,50 3,OO 3,50 4,OO 4,50 5,OO 2,oo a 1 2 3 4 5 6 7 S 9 10 11 12 13 14 48,6 44,7 41,O 38,7 34,s 31,O 28,3 26,l 24,l 1,75 2,oo 2,25 2,50 2,75 3,OO 3,25 3,50 4,OO 4,25 4,50 5,OO 3,75 4,75 Tabelle 8. Magnetisiert mit 15 Amp. Konstanten aus 1 und 4. a = 2,883. = 828,5. 15 16 17 18 6,OO 7,OO 8,OO 9,00 a beobachtet 129,s 120,4 112,7 105,l 98,6 93,O 83,9 78,9 73,O 69,s 66,9 64,l 88,s 75,9 56,l 51,O 45,3 41,2 W berechnet 129,s 112,2 98,s 93,3 120,4 105,l 88,3 83,s 79,s 76,l 72,s 69,7 66,9 64,3 55,7 49,7 43,9 39,7 Fehler 0 0 -0,5 0 + 0,2 +0,3 - 0,l +0,2 + 0,2 - 0,2 - 0,l + 0,2 - 0,5 0 - 0,4 - 1,3 - 1,4 - 1,5 Tabelle 7. Magnetisiert mit 5 Amp. 1 2 3 4 5 6 1 8 9 1,15 2,25 2,50 3,OO 3,50 4,OO 4,50 5,OO 2,oo a 48,6 44,7 41,O 38,7 34,s 31,O 28,3 26,l 24,l Tabelle 7. Magnetisiert mit 5 Amp. Tabelle 8. 1 2 3 4 5 6 7 S 9 10 11 12 13 14 1,75 2,oo 2,25 2,50 2,75 3,OO 3,25 3,50 4,OO 4,25 4,50 5,OO 3,75 4,75 Tabelle 8. Magnetisiert mit 15 Amp. Konstanten aus 1 und 4. a = 2,883. Daraus = 828,5. 15 16 17 18 6,OO 7,OO 8,OO 9,00 a beobachtet 129,s 120,4 112,7 105,l 98,6 93,O 83,9 78,9 73,O 69,s 66,9 64,l 88,s 75,9 56,l 51,O 45,3 41,2 W berechnet 129,s 112,2 98,s 93,3 120,4 105,l 88,3 83,s 79,s 76,l 72,s 69,7 66,9 64,3 55,7 49,7 43,9 39,7 Fehler 0 0 -0,5 0 + 0,2 +0,3 - 0,l +0,2 + 0,2 - 0,2 - 0,l + 0,2 - 0,5 0 - 0,4 - 1,3 - 1,4 - 1,5 Magnetisiert mit 15 Amp. Konstanten aus 1 und 4. a = 2,883. = 828,5. 568 P. Gehne. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 1,75 2,oo 2,25 2,50 2,75 3,OO 3,25 3,50 4,OO 4,25 4,50 5,OO 6,OO 7,OO 8,OO 9,00 3,75 4,75 Tabelle 9. Magnetisiert mit 25 Amp. Konstanten aus 1 und 4. a = 2,845. C = 2169,9. Spalt 11 in mm a beobachtet 342,O 318,O 296,3 276,6 258,s 244,9 233,9 219,9 208,l 199,7 192,5 183,9 176,O 169,9 145,l 131,9 109,5 11 9,0 a berecbnet 342,O 317,O 295,4 276,6 260,O 245,3 232,2 220,4 209,7 200,l 191,3 183,2 175,8 168,9 146,2 128,s 117,6 104,l Fehler 0 -1,o - 0,9 0 + 1,2 + 0,4 - 1,7 + 0,5 +1,6 +0,4 - 1,2 - O,? -0,2 - l,o -1,9 - 2,l - 5,4 -1,4 Tabelle 9. Fur j ede der drei vorstehenden Tabellen berechnete ich dann die GroBe a noch einmal und zwar unter Zugrunde- legung einer gro6eren Anzahl von Beobachtungen. Dabei er- geben sich die Mittelwerte von a fiir samtliche Slagnetisierungen als merklich gleich. Namlich: 11 ,, 15 ,, a = 2,867 MitteI n = 2,864. ,> i Magnetisiert mit 5 Amp. n = 2,862 ,, 25 ,, a = 2,864 Nach dem auf p. 560 dargelegten muSte dam sein , P7 = 7 Der mittlere Radius des magnetischen Kreises betragt 70,44 mm (vgl. p. 554); also Zi = 442,3 mm, demnach: _ - li - 154, a _ - li - 154, a dem steht entgegen pr hochstens = 58 (vgl. p. 566). dem steht entgegen pr hochstens = 58 (vgl. p. 566). 569 Perhalten permanenter Magnete usw. Die reversible Permeabilitat p,, steht also durchaus nicht mit der durch den Wert von a bedingten GroBe des inneren Widerstandes in Einklang. 1) Naheres in der Dissertation dee Verfassem p. 54. Annalen der Physik. IV. Folge. 28. 38 Daraus Um zu untersuchen, wie sich die anderen fur die Be- rechnung des inneren Widerstandes eventuell noch in Betracht kommenden GroBen zu dem Werte li/a verhalten, bestimmte ieh durch Konstruieren der Tangente an die Magnetisierungs- kurve zunachst noch die GrijBe Ich berechnete nun den inneren Widerstand des magne- tischen Kreises fur die drei auf p. 566 angegebenen Magne- tisierungen unter Zugrundelegung der Werte y, d % / d $ und pr. Da die Magnetisierung des geschlitzten Kreises nicht an allen Stellen dieselbe ist und y sowohl wie d % / d Q von dieser Magnetisierung abhiingig sind, muBte ich zunachst fur ver- schiedene Bereiche des Ringes die mittlere Induktion bei den verschiedenen Magnetisierungen bestimmen l), und dann das hierfiir giiltige p bzw. d b / d Q in Ansatz bringen; auf diese Weise erhielt ich folgende Werte fur den inneren Wider- stand wi des magnetischen KreiseB, ausgedriickt durch die aquivalente Luftstrecke in Millimetern. d B Fur p Fur Fur p, 0 )) 15 ,) ~4 = 4,86; W' =: 3,94 wi = 8 >, 1 Magnetisiert mit 5 Amp. wi = 5,98; wI = 5,30 5 ) 25 ,, Wi = 3,81; W< = 2,93 Diesen Werten gegenuber steht der fiir alle Magne- tisierungen konstante Wert Kurze Zusammenfassung der Resultate. 1. Die Variation der gesamten Kraftlinienzrthl in einern permanent magnetischen Kreise ist fur geschlitzte Toroide von relativ kleinem Querschnitt minimal, wird aber bei Toroiden gro5eren Querschnitts sehr merklich. Fur einen permanenten Magneten ist die Anzahl der gesamten Kraftlinien veranderlich, 38 38 510 P. Gehne. Perhalten permanenter Magnete usw. die magnetomotorische Kraft in einer zentralen lnduktionsrbhre in weiten Brenzen konstant. die magnetomotorische Kraft in einer zentralen lnduktionsrbhre in weiten Brenzen konstant. die magnetomotorische Kraft in einer zentralen lnduktionsrbhre in weiten Brenzen konstant. 2. Im zentralen Teil eines permanent magnetischen Kreises ist der innere Widerstand konstant. Der numerische Wert dieser WiderstandsgroBe ist aber mit keinem der Werte, die fur die Permeabilitiit in Betracht kommen konnten, in Ein- klang zu bringen. 2. Im zentralen Teil eines permanent magnetischen Kreises ist der innere Widerstand konstant. Der numerische Wert dieser WiderstandsgroBe ist aber mit keinem der Werte, die fur die Permeabilitiit in Betracht kommen konnten, in Ein- klang zu bringen. g g Die vorliegenden Untersuchungen wurden im physikalischen Institute der Universitat Halle ausgefuhrt. Es sei mir auch an dieser Stelle gestattet, meinem hoch- verehrten Lehrer, Hrn. Geh. Reg.-Rat Prof. Dr. E. Dorn, fur die freundliche Anregung zu dieser Arbeit und fur die stetige liebenswurdige Unterstiitzung und Beratung bei ihrer Sus- fiihrung meinen herzlichsten Dank ergebenst auszusprechen. (Eingegsngen 5. Dezember 1908.) 1) R. Gans, Ann. d. Phys. 27. p. 1. 1908. (Eingegsngen 5. Dezember 1908.) 1) R. Gans, Ann. d. Phys. 27. p. 1. 1908. Naohtrag. Nach AbschluB dieser Arbeit ist mir eine neue Ver- offentlichung von Hrn. R. Gans') zur Kenntnis gelangt. Er findet ebenfalls eine nicht aus Beobachtungsfehlern zu erklarende Abweichung des Wertes pT gegen alli, allerdings in geringerem MIaBe, doch in demselben Sinne wie die von mir gefundene. Hr. Gans bestimmt die GroSe p,. an Stahlstucken, die, was Dimensionen und Material betrifft, mit dem von mir auf p. 554 angefuhrten gr5Berem System ziemlich ubereinstimmen, jedoch in von Vernachlassigungen freier Weise, was mir mit Hilfe des fir diese Untersuchungen benutzten kleineren Systems nicht mtiglich war. Da der Wert von a = 7,42 mm fur mein Ringsystem 2 (vgl. p. 560) mit dem von Hrn. Gans gefundenen a = 8,312 mm einigermagen iibereinstimmt, so ist zu vermuten , da6 eine Bestimmung von p,. far dieses System ahnliche Resultate er- geben hiitte. Ein abschliebendes Urteil uber diese divergieren- den Versuchsergebnisse behalt sich Hr. Gans bis zum AbschluB weiterer Untersuchungen vor.
https://openalex.org/W3155978019	https://link.springer.com/content/pdf/10.1007/s12011-021-02716-z.pdf	English	null	A Comprehensive Review on Selenium and Its Effects on Human Health and Distribution in Middle Eastern Countries	Biological trace element research	2,021	cc-by	15,037	ERROR: type should be string, got "https://doi.org/10.1007/s12011-021-02716-z\nBiological Trace Element Research (2022) 200:971–987 https://doi.org/10.1007/s12011-021-02716-z\nBiological Trace Element Research (2022) 200:971–987 Abstract Selenium (Se) is an important microelement with numerous positive effects on human health and diseases. It is important to\nspecify that the status and consumption of Se are for a specific community as the levels of Se are extremely unpredictable\nbetween different populations and regions. Our existing paper was based on the impacts of Se on human health and disease along\nwith data on the Se levels in Middle Eastern countries. Overall, the findings of this comprehensive review show that the\nconsumption and levels of Se are inadequate in Middle Eastern nations. Such findings, together with the growing awareness\nof the importance of Se to general health, require further work primarily on creating an acceptable range of blood Se concen-\ntration or other measures to determine optimal Se consumption and, consequently, to guarantee adequate Se supplementation in\npopulations at high risk of low Se intake. Keywords Selenium . Selenoproteins . Human health . Middle East . GPx A Comprehensive Review on Selenium and Its Effects on Human\nHealth and Distribution in Middle Eastern Countries Marek Kieliszek1\n& Iqra Bano2 & Hamed Zare3 Received: 22 February 2021 /Accepted: 8 April 2021\n# The Author(s) 2021\n/ Published online: 21 April 2021 * Marek Kieliszek\nmarek_kieliszek@sggw.edu.pl; marek-kieliszek@wp.pl Introduction by Swedish scientist Jöns Jakob Berzelius in 1818 during the\nproduction of sulphuric acid [2]. Initially, it was considered to\nbe toxic to humans, until its vital function was evidenced in\nthe last decade [3]. Se plays an important role in preserving\nmany natural body functions; therefore, interest in Se research\nhas grown in many areas for public health improvement over\nthe past few decades. The biological function of Se is related\nto its incorporation through selenocysteine (SeCys) into the\nstructure of proteins important for metabolism [4]. Naturally,\nSe is present in both organic and inorganic forms. The organic\nform is the Se contained in amino acids termed as\nselenomethionine (SeMet) and SeCys, while the inorganic\nforms include selenate, selenite, selenide, and elemental Se,\nrespectively [5]. The inorganic form of Se is a major source of\ndietary Se in the human population. Animals who are raised\non Se-containing soil are a rich source of selenoproteins\n(SePs). To distribute Se across a whole country, animals\nshould be raised in fields where there is a proper concentration\nof Se in soil [6]. Due to the large differences in Se detection\nmethodologies among different laboratories, it is difficult to\ncompare Se levels in different countries [7]. It is very impor-\ntant to identify the status and intake of Se in a particular pop-\nulation as the levels of Se are highly variable between diverse\ncommunities and regions. Throughout Middle Eastern coun-\ntries, very little has been known about the level of Se intake\n[8]. Available research shows that the level of Se is highly\nheterogeneous, with some states deficient, and others Human body cells require appropriate nutrition for the main-\ntenance of homeostasis. Micronutrients, such as trace min-\nerals, antioxidants, and vitamins, are crucial for performing\nvarious regenerative processes, managing oxidative stress,\nand developing immunity against pathogens [1]. Selenium\n(Se) is one of the trace minerals that is required to maintain\nvarious functions of the body (Fig. 1). 1\nDepartment of Food Biotechnology and Microbiology, Institute of\nFood Sciences, Warsaw University of Life Sciences-SGGW,\nNowoursynowska 159C, 02-776 Warsaw, Poland Selenoproteins (SePs) SePs are considered to be proteins and perform critical biological\nroles [9] (Fig. 2). They were discovered by a Dr William\nHoekstra of the University of Wisconsin in 1973 [10]. To date,\n30 mammalian SePs have been identified from 25 genes. Structurally, they are composed of 21 amino acids [11]. During\nthe initial phases of the analysis of SePs, recognition of SePs was\nmainly dependent on experimental techniques that included pro-\ntein studies for Se through spectrometry and radioactivity analy-\nsis [12]. The largest group of selenoproteins is involved in the\nprocesses of protecting cells against the effects of reactive oxy-\ngen species (ROS). Generally, SePs perform their activities Introduction It was first discovered * Marek Kieliszek\nmarek_kieliszek@sggw.edu.pl; marek-kieliszek@wp.pl\nIqra Bano\niqrashafi05@yahoo.com\nHamed Zare\nhamed.zare1363@gmail.com\n1\nDepartment of Food Biotechnology and Microbiology, Institute of\nFood Sciences, Warsaw University of Life Sciences-SGGW,\nNowoursynowska 159C, 02-776 Warsaw, Poland\n2\nDepartment of Veterinary Physiology and Biochemistry, Shaheed\nBenazir Bhutto University of Veterinary & Animal Sciences\nSakrand, Sindh 67210, Pakistan\n3\nCellular and Molecular Research Center, Birjand University of\nMedical Sciences, Birjand, Iran 1\nDepartment of Food Biotechnology and Microbiology, Institute of\nFood Sciences, Warsaw University of Life Sciences-SGGW,\nNowoursynowska 159C, 02-776 Warsaw, Poland 2\nDepartment of Veterinary Physiology and Biochemistry, Shaheed\nBenazir Bhutto University of Veterinary & Animal Sciences\nSakrand, Sindh 67210, Pakistan 3\nCellular and Molecular Research Center, Birjand University of\nMedical Sciences, Birjand, Iran 972 Kieliszek et al. Fig. 1 Functions of selenium in various organisms Fig. 1 Functions of selenium in various organisms within cells, acting as enzymes such as deiodinases and\nthioredoxin, and also as antioxidants including glutathione per-\noxidase (GPx), which is regarded as the first animal SePs to be\ndiscovered [10]. SeP synthesis is crucial for Se maintenance and\ndisorders occurring due to disturbance of homeostasis [13]. Both\nSeCys and SeMet are crucial forms of SePs and are present\nnaturally in various sources. SeCys is found in animal tissues\nand in Se-containing proteins, whereas SeMet is obtained from\nyeast, algae, bacteria, and plants respectively [5]. Details about\nsome SePs are given below. oversufficient. The current review is based on the biological\nfunctions of Se and selenoproteins, the health benefits\nof Se, and diseases concerned with deficiency of this\ntrace mineral along with its distribution in Middle\nEastern countries including Saudi Arabia, Jordan,\nTurkey, Libya, Egypt, Iran, and Qatar. Selenoprotein-P (SelP) The\nassumption that SelP is secreted into the bloodstream, as well as\nseveral SeCys sequences that are found in its structure, indicates\nthat SePs can become a source of Se for peripheral tissues [20, 21]. Moreover, when a SelP-knockout mouse was fed a normal Se diet,\nit wasmarkedby asubstantial reductionin Selevels, particularlyin\nthe central nervous system and testes, while Se content was only\nslightly affected in the kidneys and other organs and tissues. In the Selenoprotein-P (SelP) SelP is a glycoprotein that was discovered in 1993 and is one\nof the most abundant SePs, representing some 50% of the Se\nin the blood. Research has shown that SelP plays a major role\nin selenium metabolism [5]. The reduction of systemic SelP\nexpression is associated with Se deficiency in the diet. The\nconsequence of these processes is the formation of oxidative\nstress and the occurrence of various disease states. Thus, Se A Comprehensive Review on Selenium and Its Effects on Human Health and Distribution in Middle Eastern... 973 deficiency reduces the content of SelP [14]. It was found that\nafter administration of Se, the concentration of SelP increases\nfirst and only then does the concentration of other Se-\nliver, Se was also improved by removing the SelP gene and by\nincreasing urinary excretion of Se residues [22]. Fig. 2 Examples of selenoproteins and their functions in organisms Fig. 2 Examples of selenoproteins and their functions in organisms Fig. 2 Examples of selenoproteins and their functions in organisms liver, Se was also improved by removing the SelP gene and by\nincreasing urinary excretion of Se residues [22]. deficiency reduces the content of SelP [14]. It was found that\nafter administration of Se, the concentration of SelP increases\nfirst, and only then does the concentration of other Se-\ndependent proteins increase [15]. SelP is produced in hepato-\ncytes [13]. The activity of this protein has been noted in var-\nious tissues of the body, including in plasma. It is composed of\ntwo domains: the larger N-terminal, which is responsible for\nmaintaining the redox potential in the cell, and the smaller C-\nterminal, which mediates Se transport [16]. This protein is highly\nexpressed in the testes, the brain, and liver tissues. It is considered\ntobe a marker of Se concentrationin thebody. It plays a major role\nin the transport of Se within tissues and maintains homeostasis. SelP is the first protein discovered to contain more than one Se\natom in the form of SeCys. It has also been shown that P-\nselenoprotein protects lipoproteins against oxidation by removing\nthe peroxynitrite molecule that is formed due to oxidative stress\nwithin cells as a result of the reaction between superoxide ions\nwith nitric oxide molecules at sites of inflammation [17–19]. Thioredoxin Reductase This element is found primarily in parent rocks,\nsedimentary rocks of volcanic origin, and also enters to the\nsoil together with rainfall resulting from the evaporation of\nwater from the seas and oceans. Worldwide, its content in soil\nis about 0.4 mg/kg [44, 46]. Soils with a high concentration of\nthis element are found in: North America, Ireland, Australia,\nand Israel. In turn, the areas poor in Se include, among others,\nsome provinces of China (72% of the areas in China), New\nZealand, and a large part of Europe [47]. Moreover, elevated\nSe concentrations appear in soils rich in iron compounds and\norganic matter in saline soils. The geochemical properties of\nthis element will be present in the given area. Selenides and\nselenium sulphides occur mainly in acidic, oil-rich soils with a\nhigh content of organic matter. On the other hand, in soils with\nmedium oxidation conditions and neutral pH, selenites (IV)\npredominate, which are easily soluble after combination with\nalkali metals. The environment is dominated by combinations\nof oxides with iron hydroxides (iron oxide adsorbed and fixed\nSe(IV)), which show low bioavailability in the soil. The third\ngroup are selenates (VI), which are found in well-oxidized\nalkaline soils. Selenates (VI) cannot be combined with iron\ncompounds; therefore, they are easily available. It should be\nnoted that individual forms of Se are easily transformed under\nthe influence of changes in the soil environment, and inorgan-\nic forms of Se may undergo the process of biomethylation to\nvolatile forms of Se: an example is dimethylselenide:\n(CH3)2Se. The distribution of individual compounds allows\nthe natural geochemical cycle of this element to be preserved\n[48]. The constant cycle of Se in the environment relies on the\nbiomethylation of elements by microbes and decomposition\nof the organic material present in it. As a result, volatile Se\ncompounds are synthesized that include dimethyl selenium\n(DMSe), dimethyldiselenide (DMDSe), selenium oxide\n(SeO2), and hydrogen selenide (H2Se). The Se is present in\nthe form of selenates and selenites in water [49]. In aqueous\nsolutions, Se occurs most often in the form of selenite\n(SeO2\n−3) Se (IV), selenate (SeO2\n−4) Se (VI), biselenin\n(HSeO−3), and selenic acid (H2SeO3) [50]. The concentration\nof Se is elevated in groundwater as compared to seawater. The Thioredoxin Reductase Se is a very common element on Earth and is present in the\nlithosphere, atmosphere, hydrosphere, and biosphere respec-\ntively [43]. It is released into the atmosphere via volcanic\ngases. An important factor is also the emission of this element\nto the atmosphere from industrial sources (burning coal, crude\noil) [44]. The content of Se in the atmosphere depends primar-\nily on anthropogenic activity. Human activity releases the el-\nement into the atmosphere, making up 37.5–40.6% of total\natmospheric emissions. In addition, the content of this element\nin the atmosphere in urban areas ranges from 1 to 10 ng/m3\nand mainly comes from the combustion of coal and crude oil\n[45]. Se is unevenly distributed and concentrated in the\nEarth’s crust. This element is found primarily in parent rocks,\nsedimentary rocks of volcanic origin, and also enters to the\nsoil together with rainfall resulting from the evaporation of\nwater from the seas and oceans. Worldwide, its content in soil\nis about 0.4 mg/kg [44, 46]. Soils with a high concentration of\nthis element are found in: North America, Ireland, Australia,\nand Israel. In turn, the areas poor in Se include, among others,\nsome provinces of China (72% of the areas in China), New\nZealand, and a large part of Europe [47]. Moreover, elevated\nSe concentrations appear in soils rich in iron compounds and\norganic matter in saline soils. The geochemical properties of\nthis element will be present in the given area. Selenides and\nselenium sulphides occur mainly in acidic, oil-rich soils with a\nhigh content of organic matter. On the other hand, in soils with\nmedium oxidation conditions and neutral pH, selenites (IV)\npredominate, which are easily soluble after combination with\nalkali metals. The environment is dominated by combinations\nof oxides with iron hydroxides (iron oxide adsorbed and fixed\nSe(IV)), which show low bioavailability in the soil. The third\ngroup are selenates (VI), which are found in well-oxidized\nalkaline soils. Selenates (VI) cannot be combined with iron\ncompounds; therefore, they are easily available. It should be\nnoted that individual forms of Se are easily transformed under\nthe influence of changes in the soil environment, and inorgan-\nic forms of Se may undergo the process of biomethylation to\nvolatile forms of Se: an example is dimethylselenide:\n(CH3)2Se. The distribution of individual compounds allows\nthe natural geochemical cycle of this element to be preserved\n[48]. Selenoprotein W (SepW) SepW is one of the most abundant SePs in mammals. This\nsmall 9-kDa SeP is found in the cytosol and has a high muscle\nand brain concentration [23]. Generally, SepW is required for\nmuscle metabolism in animals. However, the role of this pro-\ntein is still unknown. It has been revealed by some studies that\nits concentration was increased in animal tissues when Se was\nincreased in the diet [24]. Moreover, skeletal muscle calcifi-\ncation during white muscle disease was also prevented\nthrough Se supplementation in cattle and sheep [25]. Various studies on the relationship between SepW and human\nmuscle disease have been carried out and some researchers\ncontinue to working on this to observe SepW’s further effects\non human muscle metabolism [26]. Some studies have re-\nvealed that SepW is a part of the stress-related commu-\nnity of SePs as its expression is heavily influenced by\ndietary availability of Se [27]. Another study has re-\nvealed that when excised from rat skin, a glutathione\ncomplex was found in native SepW [28]. 974 Kieliszek et al. Thioredoxin Reductase The constant cycle of Se in the environment relies on the\nbiomethylation of elements by microbes and decomposition\nof the organic material present in it. As a result, volatile Se\ncompounds are synthesized that include dimethyl selenium\n(DMSe), dimethyldiselenide (DMDSe), selenium oxide\n(SeO2), and hydrogen selenide (H2Se). The Se is present in\nthe form of selenates and selenites in water [49]. In aqueous\nsolutions, Se occurs most often in the form of selenite\n3\n4 Thioredoxin comprises three main enzymes, including TR1,\nTR2, and TR3. They all act as intracellular antioxidant mole-\ncules by regulating redox reactions. These also act as a major\nfactor for apoptosis and DNA synthesis [29]. Thioredoxin acts\nas a key enzyme for the metabolism of Se compounds by\nproviding selenide for the synthesis of SePs [30]. Moreover,\nthis SeP is known to activate the tumor suppressor p53 gene,\nand TR1 is highly expressed in many carcinoma cells and\ntumors [31]. Furthermore, the inhibition of TR1 in cancerous\ncell lines contributed to diminishing the proliferation of cell\nand cancer progression by administering knockdown TR1\ncells into mice as compared to a control cell line [32]. TR1\ncan therefore have two conflicting functions in the develop-\nment and progression of cancer. TR1 can aid cancer preven-\ntion by preserving cell redox balance and lowering the inci-\ndence of mutations that trigger the development of tumors. TR1 is also essential for tumor progression because cancer\ncells are highly sensitive to oxidative damage [33]. TR1 has\nrecently been involved in mediating the essential role of Se in\nthe immune system and prevention of HIV infection, as well\nas its involvement in cancer [34]. Se is a very common element on Earth and is present in the\nlithosphere, atmosphere, hydrosphere, and biosphere respec-\ntively [43]. It is released into the atmosphere via volcanic\ngases. An important factor is also the emission of this element\nto the atmosphere from industrial sources (burning coal, crude\noil) [44]. The content of Se in the atmosphere depends primar-\nily on anthropogenic activity. Human activity releases the el-\nement into the atmosphere, making up 37.5–40.6% of total\natmospheric emissions. In addition, the content of this element\nin the atmosphere in urban areas ranges from 1 to 10 ng/m3\nand mainly comes from the combustion of coal and crude oil\n[45]. Se is unevenly distributed and concentrated in the\nEarth’s crust. Glutathione Peroxidase (GPx) Glutathione peroxidase (GPx) group SePs are common\nin all three domains of life (archaea, bacteria, and\neukarya) [35]. The bacteria, protozoa, fungi, and terres-\ntrial plants also contain SeCys-containing GPx sequence\nhomology. GPx carries out a variety of biological roles\nin cells, including the regulation of hydrogen peroxide\n(H2O2), hydroperoxide detoxification, and the mainte-\nnance of cell redox homeostasis [36]. GPx1 is present\nthroughout the body but highly expressed in red blood\ncells, kidneys, lungs, and liver [37]. It works as an\nantioxidant enzyme and is thought to be affected by\nSe deficiency [38]. GPx2 is present in the gastrointesti-\nnal tract and protects against oxidative stress [39]. GPx3\nis present in plasma and the extracellular fluid of the\nbody and contributes to 10–30% of the Se present in\nthe blood. It is also present in the breast, kidneys,\nlungs, liver, thyroid glands, heart, testes, and also in\nthe placenta. It reduces the production of lipid hydro-\nperoxides in plasma [40]. GPx4 is widely spread in the\nbody but strongly expressed in the testes. It resides in\nmitochondria, cytosol, and the nucleus of cells. It is\nessential for sperm maturation and motility and has a\nhighly effective impact on male fertility improvement\n[41]. GPx5 is mainly present in the olfactory epithelium\nand embryo cells [30]. The functions of GPx6, 7, and 8\nare still thought to be unknown [42]. A Comprehensive Review on Selenium and Its Effects on Human Health and Distribution in Middle Eastern... 975 Table 1 Selenium\ncontent in different\nhuman feeds\nFeeds\nSe content (mg/kg)\nBeef\n0.042–0.142\nLamb\n0.033–0.260\nChicken\n0.081–0.142\nPork\n0.032–0.198\nDairy products\n<0.001–0.11\nCereal\n0.01–0.31\nPasta\n0.01–0.10\nRice\n0.05–0.08\nPotatoes\n0.12\nTuna\n5.6\nCod\n1.5\nOnions\n<0.5\nLentils\n0.24–0.36 Se concentration in Poznań (Poland) is approximately 0.17–\n0.44 μg/L [51]. According to Stefaniak et al. [52], 8% of\n3,000 samples of tap water showed an Se content exceeding\nthe standards of the Environmental Protection Agency. The\nconcentration of Se in polluted water may vary between 400–\n700 μg/L [53]. The content of Se in water depends on the\ngeochemical environment and environmental pollution, as\nwell as the leaching of this element from the rocks. The ac-\nceptable threshold of Se in drinking water is 10 μg/L accord-\ning to World Health Organization (WHO) guidelines and the\nOffice of Environmental Health Hazard Assessment\n(OEHHA) [46]. Glutathione Peroxidase (GPx) It is worth noting that Se present in soils is\nabsorbed by plants. The absorption of this element by plants is\nconditioned by its chemical forms and concentration in soils. Some of these plants are identified to be present in China,\nwhich contains approximately 20.000 ppm Se concentration\n[54]. More than 20 seleniferous plants have been identified,\none of them being the Astragalus species [55]. Astragalus\nincludes A. bisulcatus, A. racemosus, A. praelongus,\nA. pectinatus, and A. thephorosides [54]. According to re-\nsearchers, the concentration of Se is higher in green grasses\nthan in leguminous plants [56, 57]. Plants take up Se in the\nform of selenite or selenate. Selenate is less absorbed by soil\nminerals and more easily absorbed by plants than selenite. Moreover, selenite, selenate, and selenium nanoparticles\n(SeNPs) in plant cells are reduced to organic compounds such\nas selenium amino acids (selenocysteine (SeCys)) [58]. The\nmain consumers of Se from plants are herbivores, which are\nthen consumed by humans. The excess or deficiency of this\nelement at this stage of the food chain may affect the health of\nhumans and animals. That is why it is important to monitor\nsupplementation of this element [59]. methods for determining the concentration of Se include anal-\nysis of biological samples including urine, hair, and finger-\nnails. The enteral formulas and parenteral nutrition formulas\nfor dietary Se intake have been published since 1980 by the\nfood and nutrition board of the US National Research\nCouncil. For parenteral solutions, sodium selenate is the best\nchoice to be considered. The recommended amount of Se in\nparenteral solutions for adults is 50–70 μg/day (Table 2),\nwhile for neonates and children younger than 5 years it is 2–\n3 μg/day/kg weight, and for adolescents and older children the\nrecommended dose of Se is 30–40 μg/day [65]. Nowadays, in several countries, a revolutionary technolog-\nical process focused on the processing of Se-rich food items\nincluding eggs, meat, and dairy has been successfully imple-\nmented [68]. Pork and chicken enriched with Se are vailable in\nKorea, whereas eggs fortified with Se are now on the market\nof 25 countries across the globe. When reviewing research\nstudies on the development of functional foods, it also seems\nclear that eggs enriched with Se could be used to supplement\nmicronutrient deficiencies and to sustain the body’s metabolic\nstability [69]. An egg or chicken meat with a Se content of can Se and Inflammatory Response supply 50% of the recommended daily intake for this ingredi-\nent [70]. Many research centers around the world are actively\nworking to acquire food items that are sources of Se. The most\nattractive attribute is to have the maximum bioactivity of or-\nganic shapes of Se in food items. Moreover, the use of organic\nSe supplementation of yeast origin in terms of shortage has\nmultiple beneficial effects on health [71]. The effect of Se on inflammatory responses was first identi-\nfied in diabetic rats [80]. According to some studies, it has\nbeen proved that Se can influence inflammatory responses\nby inducing inhibition of NF-kB series, which promotes the\nproduction of tumor necrosis factors and interleukins [81]. Some studies suggest that there is a sex-specific impact of\nSe on inflammatory responses, as there is an degree of sexual\ndimorphism among the distribution of Se in organs of the\nbody that affects Se status and arrangement related to body\nfunction [82]. Chronic inflammatory syndromes are usually\nlinked with a reduction in Se status and some studies have\nrevealed that patients with cystic fibrosis [39, 83], inflamma-\ntory bowel diesease, and acne [84] may have a lower status of\nSe than healthy controls. SelS (selenocysteine-containing pro-\nteins), whose secretions from liver cells and human serum\nrecognition have also been published, is one of the SePs in-\nvolved in immune responses [85, 86]. The correct supply of\nSe influences the process of transcription of the gene respon-\nsible for the synthesis of immunoglobulins (Ig), which plays a\nsignificant role in regulating the activity of the immune sys-\ntem. The Se level also influences the differentiation of macro-\nphages. In conditions of Se deficiency, more pro-\ninflammatory macrophages M1 are formed, while in condi-\ntions of Se excess, mainly antiinflammatory macrophages M2\nare formed. This model of action allows the induction of a pro-\ninflammatory cellular immune response against viral and bac-\nterial pathogens by Th1 lymphocytes [71]. It is also worth\nemphasizing that the release of various mediators of inflam-\nmatory reactions in the body may trigger the occurrence of\nvarious disease states. Lowering the activity of selenoproteins\nmay be associated with abnormalities in the functioning\nof the entire antiradical system in the body. SelS is\nregulated by inflammatory markers and extracellular\nsugar levels in liver cells [87]. Moreover, SelS has an\nantitumorigenic function in the systemic macrophages\nand brain astrocytes, decreasing stress [88]. Se and Human Health Se plays a very important role in the maintenance of\nvarious physiological processes in the human body\n[72]. It also plays a crucial role in the pathogenesis\nand pathophysiology of various disorders due to its an-\ntioxidant properties, such as oxidative stress, inflamma-\ntion, apoptosis, reproductive disorders, diabetes, thyroid\nissues, cancer, and immune responses [8]. Rising Se\ndeficiency in various parts of the world is leading to\nmany pathological disorders [73]. According to the lit-\nerature, it has been proved that people consuming a\nspecial diet (due to phenylketonuria) are especially vul-\nnerable to the adverse effects of Se deficiency [74]. Patients with liver cirrhosis, celiac disease, rheumatoid\narthritis, and other degenerative diseases have very low\nlevels of Se [74]. Furthermore, individuals exposed to\nadvanced chemotherapy, and patients who have already\nundergone radiation therapy, are susceptible to reduced\nexpression of this microelement in the system [75]. Se\ndeficiency leads to many other diseases, such as asthma\ndue to impaired GPx activity; it also promotes impaired\ncirculation, irregular heartbeats, coma, or sudden infant\ndeath syndrome [5, 6]. Keshan disease is another con-\ndition that is associated with Se deficiency. This is pe-\ndiatric cardiomyopathy that occurs mainly in young\nwomen of reproductive age and children around 2–10\nyears old [76]. While excess dietary Se commonly\ncauses food poisoning complications such as diarrhea,\nnausea, and vomiting [77, 78]. Many cases of excessive\nSe in people and livestock present in a particular geo-\ngraphic region are usually affected by large concentra-\ntions of Se in the soils. Animal grazing in fields in\nwhich the Se concentration is greater than 5 μg/g must\nbe considered hazardous to consumer health. The Se\nderivatives are characterized by various levels of toxic-\nity. Inorganic forms of Se have greater efficacy com-\npared to organic versions [79]. These findings all sug-\ngest that both excessive and insufficient levels of this\nelement have various impacts on the health of human. A range of diseases and conditions associated with Se\nintake or deficiency and involvement in pathogenesis\nare described below. The Dietary Se and Human Requirements Food enrichment with Se must be conducted in a careful and\nregulated manner to avoid serious impacts that are the inverse\nof the expected effect, since Se is among the most toxic ele-\nments in relatively small amounts, while at the very same\nbeing a vital nutrient with a significant biological role. The\ndifference between the required quantity of Se and the toxic\ndose is very small [4, 60, 61]. Other sources of Se for humans\nare dairy products, cereals, rice, and tuna (Table 1). Table 2\nRecommended dietary allowances (RDAs) for selenium intake\n[66, 67]\nGroup\nSelenium content (μg/day)\nInfants\n10–15\nChildren 1–3\n15–20\nChildren 4–8\n20–30\nChildren 9–13\n40–45\nAdults (men, women)\n55–70\nPregnant women\n60\nLactating women\n60–75 Table 2\nRecommended dietary allowances (RDAs) for selenium intake\n[66 67] The level of Se within human tissue depends on dietary\nintake, which it related to Se availability in the soil and its\ngeographical distribution [62]. One of the most critical prob-\nlems in analytical epidemiological studies based on Se is the\ndifficulty of measuring Se uptake from the diet [63]. According to some studies, the marker for adequate Se intake\nis the measurement of GPx enzyme activity, which was mea-\nsured in erythrocytes, because the activity of this enzyme is\ndirectly proportional to dietary intake of Se [64]. The other 976 Kieliszek et al. Se and Cancer It has been proved by some studies that\nSec insertion sequences (SECIS: selenocysteine insertion se-\nquence) of the open reading frame for mammalian SePs re-\nsemble that of the CD4 gene [92]. Such reading frames can\nencode amino acid sequences within potential selenoproteins. It is worth noting that the accumulation of UGA codons in\nopen reading frames for selenoproteins may refer to the exis-\ntence of specific sequences within the untranslated mRNA\nregion responsible for the insertion of selenocysteine\n(SeCys) in place of another cysteine amino acid (Cys) during\nthe biosynthesis of various proteins. In this way, the resulting\nproteins containing Se in their structure show biological activ-\nity [93]. Hence, Se is important for T-cell protein formation\nand its normal function [94]. Moreover, thioredoxin reductase\npurified from human T-cells plays an important role in reduc-\ning tumor cell growth [95]. The effects of Se status on prostate\ncancer are superior for secondary infections and diseases, sug-\ngesting an inhibitory effect on the spread of tumor [94]. The\nprocess of angiogenesis requires growth factors such as vas-\ncular endothelial cells, growth factors, and metalloproteinases\nfor the degradation of proteins residing in the extracellular\nmatrix. The metalloproteinases cause a reduction in the level\nof growth factors and proteins via methyl selenol (CH3SeH)\nprecursor [95], whereas the selenite suppresses the invasion of\nhuman fibroids by reducing expression of metalloproteinases-\n2 and metalloproteinases-9 [96]. Moreover, bladder cancer\ndevelopment is affected by the genotype of GPx1 which sug-\ngests that GPx1 is relevant to bladder cancer progression [97]. Various methods from numerous areas including China and\nthe USA have verified that low Se consumption is related to\nthe occurrence of lung, thyroid, prostate, mammary and colo-\nrectal carcinomas [98]. The association between Se and risk of\ngastric or esophageal cancer is still unclear. Meanwhile, hu-\nman experiments have been conducted with Se doses of\naround 50 mg/day along with a blend of additional supple-\nments [99, 100]. To date, various compounds of Se\nhave been tested to induce apoptosis involving various\nsignaling pathways [101]. The results revealed that Se\nsupplementation via organic Se decreased metastasis\nboth in vivo and in vitro, suggesting that Se acts as\nan antimetastatic compound by suppressing angiogene-\nsis, cancer cell migration, and invasion [102]. Se and Alzheimer’s Disease Alzheimer’s disease (AD) is a common type of dementia and\nsubsequent development of disease characterized by a pro-\ngressive loss of memory, expression, and recognition of indi-\nviduals or objects, along with more managerial, or alert be-\nhavioral changes [112]. For many years, there has been in-\ncreased interest in the role of Se in medical and neurological\ndisorders such as AD. Furthermore, it was stated in a some\nanimal studies that changes in the level of Se-containing com-\npounds affect the metabolic activity of neurotransmitters\n[113]. This seems very significant in the study of AD because\nseveral neurotransmitters are defective in this disease. However, the most important role of Se in AD is the antiox-\nidant role of distinct seleno-enzymes. Furthermore, the links\nbetween Se and AD risk factors have also been noted. Interactions between Se and apolipoprotein E (ApoE) and\npresenilin 2 are both considered to be heritable genetic factors\nfor AD [114]. Many postmortem studies involving human\nbrain autopsy and tissue biopsies from AD patients and con-\ntrol subjects have explored whether AD is related to altered\nlevels of Se [115]. Epidemiological and clinical studies on Se\nhave indicated stratified consequences in primary and second-\nary prevention of AD. Considered collectively, multiple re-\ncent studies have suggested that modification of Se is a cause Se and Cancer The prevention of cancer by the use of Se in humans, as well\nas in animals, has been indicated by various epidemiological\nstudies. A recent study conducted on 1312 cancer patients\nsupplemented with almost 200 μg Se on a daily basis showed\na decrease in tumor size in prostate cancer, lung cancer, and\ncolon cancer of up to 63%, 58%, and 46% respectively [89,\n90]. The mechanism of action is still unknown but is thought\nto be through two key Se-dependent redox systems (glutathi-\none peroxidase and thioredoxin) within cells. The specific\ncells that facilitate the extermination of toxin compounds\nand mutated cells in blood circulation are T-lymphocytes, A Comprehensive Review on Selenium and Its Effects on Human Health and Distribution in Middle Eastern... 977 Both small and large amounts of Se have been found to se-\nverely impact the cardiovascular system. A trial was under-\ntaken in Eastern USA, with a 7.6-year follow-up to evaluate\nthe impact of Se intake on CVD prevention. The occurrence of\nmyocardial infarction, complete cerebrovascular injuries, and\nCVD were evaluated, and the findings showed that there is no\noverall advantage of applying 200 μg/day of Se to avoid CVD\n[108]. In general, in the context of cardiovascular physiology,\nthe GPx family belongs to the strongest known SePs. Studies\nthat concentrated on the significance of Se deficiency in heart\ndisease development lacking infectious origin have already\nshown that correlation between an inadequate intake of Se\nand cardiac dysfunctions may arise from oxidative stress, as\nwell as its complications. Animal experiments utilizing vari-\nous medications and formulas of Se, as well as trials in GPx\nmouse models, reported an important role in neutralizing re-\nactive oxygen and nitrogen molecules, ultimately restricting\norgan injury following myocardial reperfusion. In addition to\nthe GPx subtypes, thioredoxin reductase is assumed to have\nspecific duties in the cardiovascular system by oxidizing in-\ntraspecific and extracellular signaling molecules via an influ-\nence on adaptive responses such as remodeling [109, 110]. Moreover, some experiments based on animal models have\nrevealed that Se deficiency has proved the down gradation of\nlowdensity lipoprotein (LDL) receptor, which is essential for\nregulating plasma cholesterol levels [111]. and it is thought that Se is required for activation of T-\nlymphocytes [91]. Se and Cancer It has been proved by some studies that\nSec insertion sequences (SECIS: selenocysteine insertion se-\nquence) of the open reading frame for mammalian SePs re-\nsemble that of the CD4 gene [92]. Such reading frames can\nencode amino acid sequences within potential selenoproteins. It is worth noting that the accumulation of UGA codons in\nopen reading frames for selenoproteins may refer to the exis-\ntence of specific sequences within the untranslated mRNA\nregion responsible for the insertion of selenocysteine\n(SeCys) in place of another cysteine amino acid (Cys) during\nthe biosynthesis of various proteins. In this way, the resulting\nproteins containing Se in their structure show biological activ-\nity [93]. Hence, Se is important for T-cell protein formation\nand its normal function [94]. Moreover, thioredoxin reductase\npurified from human T-cells plays an important role in reduc-\ning tumor cell growth [95]. The effects of Se status on prostate\ncancer are superior for secondary infections and diseases, sug-\ngesting an inhibitory effect on the spread of tumor [94]. The\nprocess of angiogenesis requires growth factors such as vas-\ncular endothelial cells, growth factors, and metalloproteinases\nfor the degradation of proteins residing in the extracellular\nmatrix. The metalloproteinases cause a reduction in the level\nof growth factors and proteins via methyl selenol (CH3SeH)\nprecursor [95], whereas the selenite suppresses the invasion of\nhuman fibroids by reducing expression of metalloproteinases-\n2 and metalloproteinases-9 [96]. Moreover, bladder cancer\ndevelopment is affected by the genotype of GPx1 which sug-\ngests that GPx1 is relevant to bladder cancer progression [97]. Various methods from numerous areas including China and\nthe USA have verified that low Se consumption is related to\nthe occurrence of lung, thyroid, prostate, mammary and colo-\nrectal carcinomas [98]. The association between Se and risk of\ngastric or esophageal cancer is still unclear. Meanwhile, hu-\nman experiments have been conducted with Se doses of\naround 50 mg/day along with a blend of additional supple-\nments [99, 100]. To date, various compounds of Se\nhave been tested to induce apoptosis involving various\nsignaling pathways [101]. The results revealed that Se\nsupplementation via organic Se decreased metastasis\nboth in vivo and in vitro, suggesting that Se acts as\nan antimetastatic compound by suppressing angiogene-\nsis, cancer cell migration, and invasion [102]. and it is thought that Se is required for activation of T-\nlymphocytes [91]. Se and Fertility Se is a micronutrient that is important for male and female\nreproduction [137]. The primary factor in male fertility main-\ntenance is oxidative damage to sperm cells, while Se protects\nsperm cells from this destruction and as a result acts as a key\nfactor in the maintenance of male fertility [102]. Low Se con-\ncentration may increase sperm susceptibility to free radicals,\nwhich may disturb the biochemical processes taking place in\nthe acrosome [138]. It was found that lowering the Se concen-\ntration reduces sperm motility, results in damage to the central\npart of the sperm whip, and also increases abnormalities relat-\ned to the morphological structure (mainly with an abnormal\nsperm head or folded tail) [139]. A prominent occurrence of\nfree radicals and lipid peroxidation has been observed in male\nmice fed Se-lacking and improved Se-supplemented diets\n[140, 141]. The results proved that the Se-deficient diet leads\nto a decrease in motility ability and poor-quality semen as\ncompared to increased Se in the diet [142]. Furthermore, one\nstudy showed that Se deficiency can lead to decreased pro-\nduction of spermatozoa. Generally, the total amount of Se in\ntestes can be analyzed by observing the GPx4 concentration\nwithin them [143]. In another study, 69 infertile Scottish men\nreceived Se for 3 months combined with vitamins A, C, and E. A significant increase was found in sperm motility at the end of\nthe clinical trial as compared to control patients [144]. As report-\ned by Mintziori et al. [145], Se supplementation (<200 μg/day) is\nprobably beneficial for men in terms of improving sperm motil-\nity. In other studies, it was found that supplementing the diet with Se and Cardiovascular Diseases It is believed that an antioxidant plays an important role in\ndefending against atherosclerotic and cardiovascular disease\n(CVD) events [103]. Deprivation of Se was strongly correlat-\ned with cardiomyopathy occurring in countries with signifi-\ncantly reduced Se intake [104]. Multiple studies have been\nconducted to evaluate Se’s effect on CVD risk [105–107]. 978 Kieliszek et al. of the pathogenesis of AD. However, the differences in GPx\nactivity may not be unique to AD, because no differences in\nGPx activity were found when comparing the cerebrospinal\nfluid of patient subjects with cerebrovascular disease\n[116–118]. As another example, SelM (selenoprotein M),\nwhich is a SeP has been confirmed to lead to a decreased\ncalcium stream in neurons. SelM downregulation contributed\nto a spike in cytosolic calcium levels [119]. This is notable\nbecause shifts in calcium homeostasis are claimed to be in-\nvolved in AD pathogenesis [120, 121]. Clinically, it is impor-\ntant to evaluate the consequences of Se supplementation in the\nlong term and in comparison with other possible effects to\nbuild the clinical evidence that enables an effective under-\nstanding of the impact of Se on AD prevention [112]. iodine therapy. The results suggested that Se supplementation\nleads to a reduction in thyroid-stimulating hormone (TSH)\nconcentration in healthy children [131]. Another study re-\nvealed that the concentration of Se was recuced in Graves’\norbitopathy disease as compared with patients having only\nGraves’ disease [132]. This suggests that Se deficiency may\nform an independent risk factor for Graves’ orbitopathy. Moreover, in another study, there was no significant link\nfound between Se status and severity of Graves’ orbitopathy\n[133]. Another study suggests that Se deficiency elevates risks\nfor hyperthyroidism, either from nodular goiter or from\nGraves’ disease. The alteration in thyroid hormone levels\nwas only observed in males, signifying the sexual dimorphism\nconcerning Se status and thyroid abnormalities [134]. Significantly, new innovative trials in mutually acute infec-\ntious thyroiditis (AIT) and Graves’ illness have the potential to\nchange existing scientific practice where Se is an important\nelement [135]. The determination of Se status throughout ex-\nperiments and self-medication of patients is very impor-\ntant for clarification of outcomes. Clear recommenda-\ntions for appropriate use of Se as an international guide-\nline are urgently needed [136]. Se and Type 2 Diabetes Mellitus Several in vitro and in vivo experiments have already shown\nthat Se plays a key role in glucose homeostasis regulation. Se\nhas also been found to postpone diabetes development and\nprogression. It has also been reported that Se acts as mimetic\ninsulin in the form of selenite [122–124]. Some experiments\nhave associated concentrations of plasma Se above approxi-\nmately 140 ng/mL with enhanced type 2 diabetes (T2D) risk\n[125]. These results were based on the use of T2D-consistent\ntreatment unconfirmed with glucose, insulin, or hemoglobin\n(HbA1c) circulation tests. However, this has not yet been\nverified in other randomized Se controlled studies [126]. Some animal model experiments have provided positive re-\nsults. The link between a high level of Se exposure and insulin\nresistance and glucose intolerance in late gestation and off-\nspring was found by contrasts between supranutritional\n(>190 mg Se/kg) and nutritional (<50 mg Se/kg) dietary sup-\nplementation [127]. A similar experiment with pigs has been\nfound to show a higher circulating level insulin with a diet\ncontaining 3 mg Se/kg with natural glucose relative to dietary\nlevels [128]. The consequences of T2D can be identified brief-\nly as ineffective deposition and/or insulin sensitivity. SePs are\nessential physiological antioxidants capable of exercis-\ning insulin-like properties that may excessively impede\nthe signaling of insulin [88]. Moreover, pancreatic beta\ncells demonstrate the biological plausibility of Se\nplaying a role in T2D [129]. Se Status in the Kingdom of Saudi Arabia organic Se at an amount of 0.6 and 0.9 ppm increases sperm\nmotility and vitality in Thai-native chickens [146]. Moreover,\nanother study suggested that the level of mitochondrial glutathi-\none peroxidase (mGPx4) that becomes prominent during puberty\nneutralizes free radicals created throughout a succession of redox\nreactions along inside mitochondria. Furthermore, the increased\nlevel of mGPx4, manages the appearance and arrangement of the\nmitochondrial casing by creating a transverse connection through\nitself, as well as additional proteins [147]. When the mGPx4\nconcentration declinea, the structural consistency of sperms re-\nduces, resulting in infertility. Additionally, research has revealed\nthat a low amount of mGPx4 was present in infertile males [148]. Studies in the Kingdom of Saudi Arabia (KSA) studies have\nshown that soil Se levels and food which is grown in KSA\nregions have low to poor Se concentrations [152]. According\nto the data presented by Al-Saleh [154], the content of Se in\nthe soil in KSA (in Al-Kharj, which is located in close\nproximity to Riyadh city) ranges from 0.1 to 0.11 mg/kg. The KSA government is actively making efforts to ensure\nfood safety in the Kingdom by promoting home-grown items. Even so, food imports are a major part of the local supply,\nalmost all originating from the US, whose soil Se grades ap-\npear to be higher to adequate [153]. The main factors contrib-\nuting to Se consumption in KSA are cereals and baked prod-\nucts, followed by fish and meat products, and dairy products\n[155, 156]. With the aim of helping to increase Se consump-\ntion, a study has been conducted by students of King Abdul-\nAziz University study at Jeddah on the Se content of different\nfood items [157, 158]. The major food groups tested in the\nexperiment were cereal products, legumes, and meats. Moreover, for infants residing in KSA, the consumption of\nSe is achieved through infant formulas imported from\nEurope to ensure adequate intake of Se [159]. Conversely,\nmany breast-fed infants do not get enough Se [154]. Food\nchoices may vary among cities and suburbs and various areas\nof the state where there may be variations in the accessibility\nof some imported goods. This may have a significant effect on\nSe levels observed in the KSA community [152]. Se and Thyroid Gland Although the thyroid gland requires iodine for its normal func-\ntions, recent studies have suggested that Se status in the diet\naffects the volume of the thyroid gland [130]. The first ever\nstudy on Se and thyroid metabolism was carried out in central\nAfrica with Se deficient and Se-combined diet in addition to A Comprehensive Review on Selenium and Its Effects on Human Health and Distribution in Middle Eastern... 979 The Role of Se in Pregnancy The requirement for several nutrients increases during the gesta-\ntion period due to several metabolic changes in the body [149]. The reduced Se blood concentration during pregnancy is fre-\nquently associated with reduced activity of the GPx enzyme. Several authors have demonstrated that whole-blood and red\nblood cell enzyme activity decreases throughout pregnancy due\nto Se deficiency in the blood. According to previous studies, it\nhas been proved that the concentrations of Se throughout the\ngestation period were found to be strongly reduced. GPx activity\nwas found to be substantially reduced during the first trimester\nand this low level was sustained until the third trimester, with a\nslight drop during delivery of the fetus [150, 151]. Deficiencies\nof Se in pregnant women could harm the growth of fetus, espe-\ncially it affects the nervous system of growing fetus. Moreover,\ncase-control analysis of expectant mothers found a significant\nrelationship between lower levels of Se and neural tube disabil-\nities [8]. Oxidative stress is believed to arise due to Se deficien-\ncies throughout pregnancy, leading to miscarriages, premature\nbirth, cholestasis, intrauterine retardations, preeclampsia, thyroid\ndisorders, and diabetes mellitus [152]. Se Status in the Kingdom of Saudi Arabia It is worth\nnoting that the estimated Se consumption in Saudi Arabia in a\nstudy by Al-Ahmary [158] was 75–122 μg/person/day, and\nthis level was close to the recommended level (72 μg/person/\nday). It is worth noting that in the studies conducted by Al-\nOthman et al. [156] the daily Se consumption by Saudis in the\ncity of Riyadh was greater than for Sweden (44 μg/person/\nday) or Greece (39 μg/person/day). Nevertheless, it is impor-\ntant to carry out further research aimed at determining the\nbioavailability of Se in food in order to correctly determine\nthe value of this element in the diet. Se Status in Turkey The content of Se in soil in various regions of Turkey ranges\nfrom 0.01 to 0.08 mg/kg [167]. The consumption of bread in\nTurkey by the average person resident ranges from 150 to 450\ng/day, which is almost half of the dry diet [168, 169]. Bearing\nin mind that wheat is the staple food in this country, it seems\nthat the availability of dietary Se for humans will depend on\nthe content of this element in this plant [169]. The average\nlevels of Se in white, whole grain. and maize wheat bread\nwere respectively 1.149, 1.204, and 2.023 μg/kg [168]. The\nSe concentration was measured in the colostrum and mature\nbreast milk samples of the healthy lactating women in Turkey. It was found that the Se content was below the international\nreference range set at 18.5 μg/L among all the analyzed sam-\nples during the whole lactation period [152]. Furthermore,\nvarious samples from dairy milk products were also analyzed. The study found that goat milk had the highest Se content, and\ncow milk had the lowest content of Se [8]. All dairy products\nwere analyzed in another study, where it was revealed that Se\nconcentrations varied based on the type of food. Food samples\ncontaining butter and cheese had higher concentrations,\nwhereas other products such as milk, ice cream, and yogurt\nhad almost no Se [170]. Depending on the region, the daily Se\nintake generally ranges from 4 to 84 μg [169]. Different data\nprovided by Aras et al. [171] indicate that the daily intake of Se Status in Libya Libya is located on the North African coast, bordered by the\nMediterranean Sea to the north. In Libya, groundwater is the\nprimary source of water for drinking supplies and for agriculture\n[175]. Elemental analysis of fields from two distinct agricultural\nland sectors in Libya was performed to calculate the number of\ncertain trace elements utilizing their long-lived radioactive iso-\ntopes. This showed that the amount of Se in Libyan soil is much\nlower than in other countries. The average content of Se in the\nEarth’s crust is 0.4 μg/g. The findings of the experiment indicat-\ned that Se concentrations in Libyan clay surface soil (0.16–0.62\nμg/g) were greater than in sandy soil (0.09–0.22 μg/g) [176]. The\nSe content of various Libyan food items was calculated, includ-\ning a number of types of local and imported food, such as wheat,\nrice, bread, nuts, tea, coffee, and commonly used additives such\nas black and red pepper. The results suggested that the level of Se\nin imported products, such as rice, wheat, and wheat products,\nvaries significantly with both the species of plants, as well as the\ncondition of the lands in which they were grown. Moreover, the\ncalculated and analyzed dietary Se levels tend to be lower\nthroughout the Libyan community than is recommended. Dosing of Se in Libyan diets is strongly recommended through\nintroducing Se to Libyan fields as a fertilizer application, espe-\ncially for cereal schemes [177]. Se Status in Jordan The\napproximate daily average dose and noncarcinogenic hazard\nmeasurement values for Se exposure were below the level of\nconcern for negative health effects [165]. In conclusion, it\nshould be noted that the local grain harvest in Jordan is largely\ninsufficient to meet demand. That is why Jordan is dependent\non food imports. In addition, the presented data may not re-\nflect the actual nutritional status of the population of this coun-\ntry with regard to this trace element (Se) [166]; therefore, it is\nworth carrying out further research in this direction to deter-\nmine the level of this element in Jordan. Se is 20–53 μg/day. In the case of the estimated Se intake in\nchildren, this was reported as 30–40 μg/day [172]. Most stud-\nies from Turkey have shown that the community has an sub-\noptimal Se level [173, 174]. However, other studies docu-\nmented concentrations of plasma [70] and serum Se slightly\nabove 120 μg/L [173]. Se Status in Jordan Se status in Middle Eastern countries is widely variable, as Se\nis found less in some countries and more in others. This var-\niability is apparent even among provinces in the same country. These variations can be related to varying concentrations of\nthe Se in the soil where food is grown. In this section of the\nreview paper, we summarize the status of Se and its intake\namong Middle Eastern countries based on the available liter-\nature. Here, it should be noted that the majority of studies that\nassessed the concentrations of Se in biological samples were\ncase-controlled with small sample sizes, and cannot be gener-\nalized to the whole population. The experiments based on Se\naround the Middle East have various findings [153]. There are currently no scientific data on the Se content of soil\nin Jordan. Only the content of Se in the water has been deter-\nmined. A groundwater survey was carried out to assess Se\nlevels in several aquifers at Amman Zarqa Basin, which is\nregareded as the most populated and industrialized basin in\nJordan. Se levels ranged from 0.09 to 0.74 mg/L across dif-\nferent aquifers, with an average of 24 μg/L, which exceeds the\nWHO recommended drinking water threshold [160]. Another\npublication by Mistry et al. [161] states that the average con-\ntent of Se in water is 0.308 mg/L. Thus, it can be noted that the\ncontent of this element in soil and food grown in these areas 980 Kieliszek et al. may be very low. A later case-control analysis in patients with\ncolorectal cancer tested Se consumption (38.75 ±11.42 μg/\nday) which was significantly lower than the control (59.26\n±8.91 μg/day). It should be noted that the recommended daily\nallowances (RDA) for Se should be at the level of 60–70 μg/\nday [162]. Concentrations of Se have, however, been exam-\nined in two studies using samples of blood and hair [163]. Blood concentrations of about 187 μg/L were considered to\nbe comparatively large in nonsmokers [164]. According to the\nliterature, it has been revealed that in Jordan the assessment of\nseasonal trends in distributions of Se displayed greater levels\nduring the dry season. The strategy including all basic of po-\ntential sources and factors leading to the water content of Se\nhas been reviewed. Serious adverse risk analysis and exposure\namong residents using spring water was also estimated. Se Status in Junhuriyah Misr-Al-Arabiya\n(Egypt) Se is not mentioned in the Egyptian food formulation table. Therefore, limited studies have been undertaken to evaluate Se\nin various food groups, to support a formal decision on\nEgyptians’ dietary Se intake [178]. Consumption of 8.3 mg/day\namong healthy children in Egypt has been reported [179]. Case-\ncontrol studies were performed primarily in children living in\nEgypt and Se levels of 65–83 μg/L were documented [8]. It\nshould be noted that skilful research and processes aimed at\ndetermining the enrichment of this element in the diet will likely\nincrease Se supplementation by people living in Egypt. To sum\nup, the condition of proper human nutrition is complete coverage\nof the body’s energy requirements and individual nutrients need-\ned for physical and mental development and maintaining full\nhealth. Appropriate balancing of Se in the diet can help to min-\nimize the occurrence of various disease states. A Comprehensive Review on Selenium and Its Effects on Human Health and Distribution in Middle Eastern... 981 Declarations Competing Interests\nThe authors declare no competing interests. Open Access This article is licensed under a Creative Commons\nAttribution 4.0 International License, which permits use, sharing, adap-\ntation, distribution and reproduction in any medium or format, as long as\nyou give appropriate credit to the original author(s) and the source, pro-\nvide a link to the Creative Commons licence, and indicate if changes were\nmade. The images or other third party material in this article are included\nin the article's Creative Commons licence, unless indicated otherwise in a\ncredit line to the material. If material is not included in the article's\nCreative Commons licence and your intended use is not permitted by\nstatutory regulation or exceeds the permitted use, you will need to obtain\npermission directly from the copyright holder. To view a copy of this\nlicence, visit http://creativecommons.org/licenses/by/4.0/. Se Status in Qatar (Dawlat Qatar) In Qatar, there have been no direct studies to determine\nSe intake among the Qatari population. The concentra-\ntion of Se in soils is low (0.12–0.77 mg/kg) [186]. According to Qatar General Electricity & Water\nCompany (Kahramaa), Se is not believed to be present\nin the water system in Qatar [187]. However, studies\npresented by Sharm [186] showed that the selenium\ncontent in groundwater is from 0.6 to 80 μg/L [186]. Besides, in Qatar’s primary staple food plant, a survey\nof Se in imported rice concluded that rice makes up\nmore than 100% of the reference nutritive intake\n(RNI) of Se, which was 30 μg/day for Qatari citizens. However, for foreign residents of Qatar (more than 80%\nof the population) who ingested much less rice, the\npercentages varied with sex and rice variety, and all\nwere below 100% of RNI Se. Further research included\nrice-based infant cereals in Qatar, which were found to\nprovide approximately 63% of RNI Se based on the\ndaily portion recommended [8]. This may mean that\nthe use of rice in Qatar contributes greatly to daily\nintake of Se. Even so, Se status was not evaluated in\nany of the several available studies in Qatar. Se Status in Iran The content of soil-Se in Iran ranges from 0.04 to 0.45 mg/kg\n[180]. Considering that rice is one of the most important foods\nin Iran, it may be of interest to determine the content of Se in\nthis product. Rahimzadeh-Barzoki et al. [181] investigated the\nassociation between Se concentration in rice and the rate of\nesophageal malignancy in Golestan province, Iran. They mea-\nsured the Se content of various samples of rice. The mean Se\nlevel in rice samples was 0.229 mg/kg. Moreover, Se levels in\nIranian rice samples were significantly higher than in other\ncountries [178] (0.020 mg/kg and 0.05 mg/kg in Italy and\nKorea, respectively) [182]. Extensive studies on Se content\nin different groups including adolescents, adults, and the el-\nderly have been conducted in Iran [178, 180]. The intake in\nadults and children was found to be sufficient, whereas post-\nmenopausal women had a significantly lower intake than the\nRDA, which is 55 μg/day [180]. Another study revealed that\nthe serum selenium concentration ranged from 58 to 123 μg/L\nin infants, adults, and expectant mothers [183]. Mirzaeian\net al. [184] assess Se intakes in female students in Isfahan,\nIran. Food intake analysis indicated that the amount of Se\nwas 54.5 μg/day, which is not significantly different from\nthe recommended value (55 μg/day). According to other stud-\nies, it can be said that Se intake was found to be adequate in\nIranian children and adults [8, 152, 185]. Se is an important trace mineral for successful functionality of\nhuman and animal species. Unlike many other trace elements,\nit is an element with a very limited quantitative range of con-\ncentrations between deficiency and physiological status, as\nwell as toxic concentrations. 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https://openalex.org/W2469215166	https://www.nature.com/articles/ncomms12080.pdf	English	null	IL-13 from intraepithelial lymphocytes regulates tissue homeostasis and protects against carcinogenesis in the skin	Nature communications	2,016	cc-by	15,329	ARTICLE Received 18 Jan 2016 \| Accepted 26 May 2016 \| Published 30 Jun 2016 Received 18 Jan 2016 \| Accepted 26 May 2016 \| Published 30 Jun 2016 Results Ski IE Skin IELs are potent producers of IL-13. The importance of tissue-speciﬁc IELs for host protection is evident by the marked increase in skin tumour susceptibility of mice lacking only Vg5Vd1 þ skin IELs9. The tumour protection conferred by gd T cells has primarily been attributed to their cytotoxicity and them being an early source of interferon-g (IFN-g) (refs 15,16). However, we found that, when skin IELs were activated in situ by a variety of environmental stressors, their principal stress response was to produce type-2 cytokines, primarily IL-13 (Fig. 1). In the resting skin, IELs are morphologically very dendritic and use these dendrites to form contact points with the ECs17 (Supplementary Fig. 1). Upon a mild topical physical insult, or skin exposure to chemicals such as 12-O-tetradecanoylphorbol-13-acetate (TPA) or the carcinogen 7,12-dimethylbenz[a]anthracene (DMBA), IELs retracted their dendrites and rounded up their cell bodies (Supplementary Fig. 1), which parallels with IEL activation9. At the same time, IL- 13 was upregulated in the epidermal tissue. This was a conserved response to stress and was seen both after exposure to physical stressors, such as tape-stripping and shaving, or following exposure to ultraviolet irradiation (UV) and chemicals such as TPA or DMBA (Fig. 1a)—and even after transgenic upregulation of NKG2D ligands on skin ECs12. The vast majority of freshly isolated skin IELs stimulated ex vivo also produced potent amounts of IL-13 protein within just a few hours, while they did not produce IFN-g or IL-17 (Fig. 1b,c). Importantly, the IELs were the only cells in the epidermis to produce IL-13 (Fig. 1d). This IL-13 signature was unique to the Vg5Vd1 þ skin IELs and not a universal gd T-cell phenomenon, as systemic gd T cells isolated from the skin draining lymph node or spleen did not show this IL-13 bias (Fig. 1e; supplementary Fig. 2). Using IL-13- egfp reporter mice, we found that Vg5Vd1 þ epidermal IELs were in fact positive for IL-13 already at steady state in the resting skin, and that IL-13 expression was further upregulated following in vivo topical exposure to TPA (Fig. 1f,g) and other environmental challenges (Supplementary Fig. 3). In contrast, Vg5 dermal gd T cells were negative for IL-13-egfp (Fig. 1f,g). Hence, the epidermal IELs constitutively produce IL-13 at steady state, show a distinctive and conserved IL-13 signature when responding to environmental stress and are the only cells in the epidermis to produce IL-13. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms12080 NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms12080 I I mmune surveillance refers to the capacity of the immune system to sense cellular dysregulation and respond to restore homeostasis. This continued ‘quality control’ mechanism has most commonly been studied in relation to cancer1–4. However, the concept of immune surveillance can also be more broadly applied to non-malignant pathologies and homeostatic tissue control5. Here we investigate the importance of IEL–EC cross-talk and type-2 immunity in skin homeostasis. We show that an important part of LSS is rapid IL-13 production by IELs, and that this provides a molecular bridge between the IELs and ECs. IEL-derived IL-13 has prominent effects on skin ECs, enabling a canonical EC response to stress with the release of TSLP and IL-33, and accelerating the rate of EC movement through the epidermis. Mice lacking IL-13, or canonical IEL, have evidence of stress in the skin, with enhanced transepidermal water loss (TEWL) and defects in the restoration of skin barrier integrity after insult. In addition, mice lacking IL-13 are more susceptible to cutaneous carcinogenesis. Hence, LSS-induced type-2 immunity is part of an early ‘allergic’ host defence mechanism regulating tissue homeostasis and protecting against carcinogenesis. The epithelial barriers of our body surface tissues are continually exposed to environmental challenges and must respond appropriately to maintain tissue integrity. Epithelial cells (ECs), which line body surface tissues, are dynamic and versatile cells, and evidence indicates that they are important drivers of immune surveillance6. In close association with ECs are the specialized tissue-resident T cells known as intraepithelial lymphocytes (IELs). IELs constitute a large, but somewhat enigmatic, population of T lymphocytes. They carry recombination activating gene (RAG)-dependent rearranged T-cell receptors (TCRs), yet have limited TCR diversity and are mainly major histocompatibility complex non-restricted cells. They express innate receptors that enable reaction to stress antigens with rapid ‘innate-like’ response kinetics7. The murine skin contains a unique subset of gd TCR þ IELs, called dendritic epidermal T cells, that exclusively carry a Vg5Vd1 TCR and constitutively express stress-sensing receptors such as NKG2D. NKG2D ligands are induced on stressed ECs, are expressed by most epithelial tumours and the NKG2D pathway is strongly associated with anti-tumour responses in both humans and mice8. We have previously shown that skin IELs directly recognize and respond to alterations in autologous stress antigens on local ECs (keratinocytes (KCs)), including ligands for the NKG2D receptor9. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms12080 This rapid afferent sensing of stressed ECs probably has a crucial role in the early detection of pre-malignant cells and has been termed ‘lymphoid stress surveillance’ (LSS)10,11. Indeed, the absence of canonical skin IELs confers a signiﬁcant increase in the susceptibility to skin carcinogenesis9. Curiously, when tumour-protective skin-resident IELs are activated by stressed ECs in the LSS response, a dominant Th2-biased downstream response is triggered with large amounts of interleukin (IL)-13 and IgE being produced. This stress-induced type-2 immune response is dependent on canonical IEL recognition of stressed ECs via the NKG2D pathway12. Although Th2 immunity has traditionally been thought to impair host tumour eradication, the surprising association between a stress sensor such as NKG2D and induction of type-2 immunity necessitates investigation into the role of early type-2 immunity in cancer immune surveillance. IL-13 from intraepithelial lymphocytes regulates tissue homeostasis and protects against carcinogenesis in the skin m Dalessandri1, Greg Crawford1, Mark Hayes1, Rocio Castro Seoane1 & Jessica Strid1 Tim Dalessandri1, Greg Crawford1, Mark Hayes1, Rocio Castro Seoane1 & Jessica Strid1 The skin is under constant renewal and exposure to environmental challenges. How homeostasis is maintained alongside protective mechanisms against damage is unclear. Among the basal epithelial cells (ECs) is a population of resident intraepithelial lymphocytes (IELs) that provide host-protective immune surveillance. Here we show that IELs cross-communicate with ECs via the production of IL-13. Skin ECs are activated by IEL-derived IL-13, enabling a canonical EC stress response. In the absence of IL-13, or canonical IEL, the skin has decreased ability to repair its barrier and increased susceptibility to cutaneous carcinogenesis. IL-13 controls the rate of EC movement through the epidermis, which might explain the importance of IL-13 for epidermal integrity and its suppressive effect on skin carcinogenesis. These ﬁndings show that IL-13 acts as a molecular bridge between IELs and ECs, and reveal a critical host-defensive role for type-2 immunity in regulating EC tissue homeostasis and carcinogenesis. 1 Division of Immunology and Inﬂammation, Department of Medicine, Imperial College London, London W12 0NN, UK. Correspondence and requests for materials should be addressed to J.S. (email: j.strid@imperial.ac.uk). 1 Division of Immunology and Inﬂammation, Department of Medicine, Imperial College London, London W12 0NN, UK. materials should be addressed to J.S. (email: j.strid@imperial.ac.uk). 1 NATURE COMMUNICATIONS \| 7:12080 \| DOI: 10.1038/ncomms12080 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| 7:12080 \| DOI: 10.1038/ncomms12080 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms12080 As controls, we generated chimeric mice in which IELs were IL-13 deﬁcient, but other haematopoietic cells were IL-13 sufﬁcient, by reconstituting irradiated IL-13 / mice with WT BM (WT-IL-13 / ), as well as WT mice reconstituted with WT BM (WT-WT) and IL-13 / mice reconstituted with IL-13 / BM (IL-13 / -IL-13 / ). The chimeric mice all had similar numbers of canonical IELs and these were egfp þ in IL-13 / -IL-13 / and WT- IL-13 / chimeras in which radioresistant host IELs were attempting to produce IL-13, but not in chimeras containing WT IELs (Supplementary Fig. 5D,E). Production of IL-13 was upregulated further on stimulation of IELs with PMA and ionomycin (Supplementary Fig. 5E). Conversely, egfp þ cells were only found in other lymphoid tissues in chimeric mice reconstituted with IL-13 / BM. Topical exposure of the chimeric mice to DMBA showed that mice that lacked IL-13 in the hematopoietic system, but had IL-13-sufﬁcient IELs, had a normal KC response to stress, as shown by the induction of TSLP and IL-33. In contrast, KCs from mice with IL-13-deﬁcient IELs (but IL-13-sufﬁcient hematopoietic cells) did not respond normally to DMBA-induced stress (Fig. 2i,j). We conclude that An important mechanism of modulating IL-13 responses may be through the regulation of its receptors, and indeed following topical skin abrasion (Supplementary Fig. 4A,B) or exposure to DMBA (Supplementary Fig. 4C,D) both IL-4Ra1 and IL-13Ra1 were further upregulated. The regulation of the IL-13 receptors appeared to be independent of IL-13, as a similar upregulation on stressed KCs in vivo was seen in mice deﬁcient in IL-13 (Supplementary Fig. 4C,D). The IL-13Rs on KCs were active as addition of recombinant (r)IL-13 to primary KCs in vitro induced further upregulation of IL-4Ra1 and particularly of the ‘decoy’ receptor IL-13Ra2 (Supplementary Fig. 4E–G), as well as production of TSLP, IL-1a and TNFa (Fig. 2c–e). In parallel, IL-13 was essential for the canonical KC stress response in vivo, as IL-13-deﬁcient mice exposed topically to the carcinogen DMBA did not show the induction of TSLP, IL-33 or caspase-3 seen in wild-type (WT) mice (Fig. 2f–h). To determine whether IELs were the source of this IL-13, we exploited the fact that skin IELs are radioresistant (Supplementary Fig. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms12080 NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms12080 a Untreated Tape-stripping Shaving UV TPA DMBA 0.0000 0.0005 0.0010 0.0015 Relative expression of IL-13 Vγ5 Vγ5 Isotype IL-13 IL-13 CD45 b c Epidermis LN Spleen 0 20 40 60 80 % Cytokine + γδ T cells IL-13 IFNγ e IL-13-egfp Vγ5+ Vγ5– WT Vγ5+ IL13egfp Vγ5+ 0 500 1,000 1,500 2,000 MFI of IL-13-egfp WT Vγ5– IL13egfp Vγ5– f g d IFNγ IL-17 – + – + – + – + TPA ** * ** Events 105 105 106 104 104 103 103 102 –102 0 105 104 103 102 102 –102 0 105 104 103 –1020 0 105 105 106 104 104 103 103 102 –102 0 0 105 104 103 0 105 104 103 0 IL-13+ 4.74 IL13+ 81.6 105 102 104 103 0 105 102 104 103 0 1.08 97.1 0 1.79 Figure 1 \| Skin IELs are potent producers of IL-13. (a) Quantitative RT–PCR analysis of IL-13 mRNA in the epidermis isolated 4 h after the indicated topical stress. Data are expressed as mean±1 s.e.m. relative to the control gene cyclophylin (n ¼ 3–6 per condition). (b–d) Intracellular protein staining in Vg5 þ skin IELs following 4 h ex vivo stimulation with PMA/iono of (b–d) IL-13 and (c) IFN-g and IL-17. In d, CD45hi Vg5 þ skin IELs (red) are shown with CD45mid Langerin þ LCs (blue) and CD45 KCs (black). Representative FACS plots shown. (e) gd T cells isolated from the epidermis, LN or spleen compared for intracellular IL-13 and INFg production following 4 h ex vivo stimulation with PMA/Iono (n ¼ 3). (a–e) data from WT FVB mice. (f) Vg5 þ and Vg5 TCRgd þ T cells isolated from naive skin of BALB/c mice (grey) and IL-13egfp reporter mice (black lines) and (g) 24 h after topical exposure to TPA. (f) Shows histograms of the gfp þ gd Tcells and (g) the MFI of IL-13egfp before and after TPA challenge (n ¼ 3 and 4). Statistical signiﬁcance of difference was determined using Student’s t-test for unpaired data with Po0.001 and *Po0.0001. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms12080 a Untreated Tape-stripping Shaving UV TPA DMBA 0.0000 0.0005 0.0010 0.0015 Relative expression of IL-13 * ** Vγ5 Isotype IL 13 b 105 105 106 104 104 103 103 102 –102 0 0 IL-13+ 4.74 Vγ5 Vγ5 Isotype IL-13 b 105 105 106 104 104 103 103 102 –102 0 0 105 105 106 104 104 103 103 102 –102 0 0 IL-13+ 4.74 IL13+ 81.6 b U Tape c IFNγ IL-17 105 104 103 102 –102 0 105 104 103 0 1.08 97.1 0 1.79 IL-13 CD45 d 102 105 104 103 –1020 105 104 103 0 IL 13 CD45 Epidermis LN Spleen 0 20 40 60 80 % Cytokine + γδ T cells IL-13 IFNγ e d 102 105 104 103 –1020 105 104 103 0 d d c e WT Vγ5+ IL13egfp Vγ5+ 0 500 1,000 1,500 2,000 MFI of IL-13-egfp WT Vγ5– IL13egfp Vγ5– g – + – + – + – + TPA f f IL-13-egfp Vγ5+ Vγ5– f Events 105 102 104 103 0 105 102 104 103 0 g Figure 1 \| Skin IELs are potent producers of IL-13. (a) Quantitative RT–PCR analysis of IL-13 mRNA in the epidermis isolated 4 h after the indicated topical stress. Data are expressed as mean±1 s.e.m. relative to the control gene cyclophylin (n ¼ 3–6 per condition). (b–d) Intracellular protein staining in Vg5 þ skin IELs following 4 h ex vivo stimulation with PMA/iono of (b–d) IL-13 and (c) IFN-g and IL-17. In d, CD45hi Vg5 þ skin IELs (red) are shown with CD45mid Langerin þ LCs (blue) and CD45 KCs (black). Representative FACS plots shown. (e) gd T cells isolated from the epidermis, LN or spleen compared for intracellular IL-13 and INFg production following 4 h ex vivo stimulation with PMA/Iono (n ¼ 3). (a–e) data from WT FVB mice. (f) Vg5 þ and Vg5 TCRgd þ T cells isolated from naive skin of BALB/c mice (grey) and IL-13egfp reporter mice (black lines) and (g) 24 h after topical exposure to TPA. (f) Shows histograms of the gfp þ gd Tcells and (g) the MFI of IL-13egfp before and after TPA challenge (n ¼ 3 and 4). Statistical signiﬁcance of difference was determined using Student’s t-test for unpaired data with Po0.001 and *Po0.0001. (IL-13egfp/egfp-WT); for brevity, IL-13egfp/egfp mice are termed IL-13 / henceforth. Results Ski IE yp y Stressed ECs promptly release many cytokines; among the most robustly expressed are IL-25, IL-33 and thymic stromal lymphopoietin (TSLP), all of which can drive type-2 immune responses. This propensity of damaged epithelial tissues to induce type-2 immunity may underlie the high frequency of allergic and atopic disease at the skin and mucosal surfaces. However, despite the intense interest in this area, the cellular and molecular basis of how type-2 immunity is linked with EC dysregulation and barrier disruption is not fully understood, nor is the functional role of this type of immunity for EC homeostasis or immune surveillance fully elucidated. The relationship between allergic disease and cancer has been long-debated, but the biological nature of this association is unclear. Overviews of the epidemiology literature show both potent inverse and positive associations. This divergence highlights the complexity of the underlying interac- tions as well as reﬂects the heterogeneity of these diseases. Intriguingly, inverse associations are more common for tissues that interface with the external environment, such as the skin13,14. Nonetheless, molecular mechanism(s) for how atopy may translate into a distinct functional advantage against EC carcinogenesis have not been described. IEL-derived IL-13 activates skin ECs. IL-13 mediates its effects by interacting with a complex receptor system comprised of IL-4Ra and two IL-13-binding proteins, IL-13Ra1 and IL-13Ra2 (ref. 18). At steady state in the resting skin, all the basal keratin 5 þ KCs expressed the main IL-13Ra1 (Fig. 2a,b) and IL-4Ra1 and IL-13Ra2 were also expressed by KCs (Supplementary Fig. 4). NATURE COMMUNICATIONS \| 7:12080 \| DOI: 10.1038/ncomms12080 \| www.nature.com/naturecommunications 2 ARTICLE NATURE COMMUNICATIONS \| 7:12080 \| DOI: 10.1038/ncomms12080 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms12080 5) and generated bone marrow (BM) chimeras, in which IELs were the only haematopoietic cells in the skin that could produce IL-13. This was achieved by reconstituting irradiated WT mice with BM from mice in which both IL-13 alleles had been replaced by egfp NATURE COMMUNICATIONS \| 7:12080 \| DOI: 10.1038/ncomms12080 \| www.nature.com/naturecommunications 3 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms12080 Keratin 5 CD45 a b Keratin 5 IL-13Rα1 0.00 0.01 0.02 0.03 0.04 0.05 Relative expression of TSLP 0.00 0.02 0.04 0.06 0.08 0.10 Relative expression of IL-1α UT + rIL-13 0.0000 0.0005 0.0010 Relative expression of TNF-α c d e 0 72 0.000 0.005 0.010 0.015 Relative expression of TSLP Time (h) WT IL-13–/– f 0 72 0.000 0.005 0.010 Time (h) Relative expression of IL-33 g 0 72 0.000 0.001 0.002 0.003 0.004 Relative expression of casp 3 Time (h) h 0.000 0.005 0.010 0.015 Time (h) Relative expression of TSLP WT->WT IL-13–/–->IL-13–/– IL-13–/–->WT WT->IL-13–/– 0 24 72 0 24 72 0 24 72 0 24 72 0.00 0.02 0.04 Time (h) Relative expression of IL-33 0 24 72 0 24 72 0 24 72 0 24 72 i j * * * * * ** *** UT + rIL-13 UT + rIL-13 Figure 2 \| IEL-derived IL-13 activates skin EC. (a,b) Epidermal cell suspensions from resting WT skin stained for CD45, keratin 5 and IL-13Ra1. Representative examples shown. (c–e) Primary neonatal KCs cultured in vitro with/without 20 ng ml 1 rIL-13 were assessed for expression of (c) TS (d) IL-1a and (e) TNFa (n ¼ 6). UT, untreated. (a–e) data from WT FVB mice. (f–h) WTand IL-13 / mice were treated topically in vivo with DMBA o and freshly isolated epidermis analysed by qRT–PCR 72 h later for expression of the genes (f) TSLP, (g) IL-33 and (h) caspase-3. (i,j) Fully reconstituted chimera mice were treated with a single skin exposure of DMBA (8 weeks after BM transplant) and isolated epidermis analysed for (i) TSLP and (j) IL expression at the indicated time points (n ¼ 4 per time point). IL-13 / -WT indicates that the donor is IL-13 / and the host WT (IL-13-sufﬁci IELs); WT-IL-13 / indicates that the donor is WTand the host IL-13 / (IL-13-deﬁcient IELs). Data in c–j were done by qRT–PCR and are expresse mean±1 s.e.m. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms12080 relative to the control gene cyclophylin. WT mice are shown in black bars and IL-13 / mice in orange bars. (f–j) Data from mice BALB/c background. Statistical signiﬁcance of difference between experimental groups was determined using Student’s t-test for unpaired data with Po0.05, Po0.01, Po0.001 and Po0.0001. Keratin 5 CD45 a b Keratin 5 IL-13Rα1 0.00 0.01 0.02 0.03 0.04 0.05 Relative expression of TSLP 0.00 0.02 0.04 0.06 0.08 0.10 Relative expression of IL-1α UT + rIL-13 0.0000 0.0005 0.0010 Relative expression of TNF-α c d e 0 72 0.000 0.005 0.010 0.015 Relative expression of TSLP Time (h) WT IL-13–/– f 0 72 0.000 0.005 0.010 Time (h) Relative expression of IL-33 g 0 72 0.000 0.001 0.002 0.003 0.004 Relative expression of casp 3 Time (h) h i j * * * * * ** ** UT + rIL-13 UT + rIL-13 Keratin 5 CD45 a b Keratin 5 IL-13Rα1 b a UT + rIL-13 0.0000 0.0005 0.0010 Relative expression of TNF-α e * 0.00 0.02 0.04 0.06 0.08 0.10 Relative expression of IL-1α d * UT + rIL-13 0.00 0.01 0.02 0.03 0.04 0.05 Relative expression of TSLP c ** UT + rIL-13 d c UT 0 72 0.000 0.005 0.010 0.015 Relative expression of TSLP Time (h) WT IL-13–/– f 0 72 0.000 0.005 0.010 Time (h) Relative expression of IL-33 g 0 72 0.000 0.001 0.002 0.003 0.004 Relative expression of casp 3 Time (h) h * * * 0 72 0.000 0.005 0.010 Time (h) Relative expression of IL-33 g 0 72 0.000 0.001 0.002 0.003 0.004 Relative expression of casp 3 Time (h) h * * g 0.000 0.005 0.010 0.015 Time (h) Relative expression of TSLP WT->WT IL-13–/–->IL-13–/– IL-13–/–->WT WT->IL-13–/– 0 24 72 0 24 72 0 24 72 0 24 72 i ** 0.00 0.02 0.04 Time (h) Relative expression of IL-33 0 24 72 0 24 72 0 24 72 0 24 72 j * i j Time (h) tivates skin EC. (a,b) Epidermal cell suspensions from resting WT skin stained for CD45, keratin 5 and IL-13Ra1. 1 Figure 2 \| IEL-derived IL-13 activates skin EC. (a,b) Epidermal cell suspensions from resting WT skin stained for CD45, keratin 5 and IL-13Ra1. Representative examples shown. NATURE COMMUNICATIONS \| 7:12080 \| DOI: 10.1038/ncomms12080 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| 7:12080 \| DOI: 10.1038/ncomms12080 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms12080 NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms12080 ARTICLE WT Tcrd–/– 0.00 0.01 0.02 0.03 Relative expression of Rae-1 0.00 0.05 0.10 0.15 Relative expression of Rae-1 WT Tcrd–/– 0 2 4 6 8 10 TEWL (g hm–2) TEWL (g hm–2) WT IL-13–/– 0 2 4 6 8 10 0 20 40 60 80 100 0 20 40 60 80 100 Time (h) WT WT Tcrd–/– 50 60 70 80 90 100 TEWL (g hm–2) TEWL (g hm–2) % of max TEWL 0 20 40 60 80 100 120 0 20 40 60 80 100 Time (h) % of max TEWL IL-13–/– 60 70 80 90 100 110 0 20 40 60 80 100 0 20 40 60 80 100 % of max TEWL % of max TEWL Time (h) 0 20 40 60 80 100 0 20 40 60 80 100 Time (h) b a d c f e h g * ** ** * P=0.0011 P=0.0480 P=NS P = NS WT WT IL-13–/– IL-13–/– IL-13–/– WT WT IL-13–/– Donor: Host: Figure 3 \| IELs and IL-13 maintain tissue health and restore integrity after insult. Rae-1 expression in the epidermis of naive (a) FVB WT (n ¼ 5), FVB Tcrd / (n ¼ 5) and (B) WT BALB/c /IL-13 / BM chimeras (n ¼ 4 per group) was analysed by qRT–PCR. Data are expressed as mean±1 s.e.m. relative to the control gene cyclophilin. In b, IL-13 / donor to WT have IL-13-sufﬁcient IELs; WT donor to IL-13 / host have IL-13-deﬁcient IELs. (c–h) TEWL was analysed as a measure of barrier integrity using a tewameter probe assessing water evaporation rate and reported as g hm 2 (where g ¼ water loss in grams, h ¼ time in hours, m2 ¼ metres squared). TEWL was measured in naive (c) FVB WTand Tcrd / mice (n ¼ 20 per group) as well as (d) BALB/c WTand IL-13 / mice (n ¼ 25–27 per group). (e,f) The dorsal ear skin was abraded by tape-stripping (6 ) and TEWL measured just after tape-stripping and at indicated time points the following days in (e) FVB WT and Tcrd / and (f) BALB/c WT and IL-13 / mice (n ¼ 10 in all groups). NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms12080 Statistical signiﬁcance in a–d and inset graphs in e–f were determined using Student’s t-test for unpaired data with Po0.01, *Po0.001 and *Po0.0001. In e–h, statistical signiﬁcance of difference between experimental groups was assessed over time using linear regression. NS, not signiﬁcant. (Fig. 3c,d). Moreover, when the skin of mice lacking canonical IELs or IL-13 was challenged by removing the stratum corneum by tape-stripping, an increased degree of water loss was observed, suggesting a worse barrier function and a poorer tolerance to damage (Fig. 3e,f, inserts). More strikingly, mice deﬁcient in IELs or IL-13 showed a signiﬁcantly impaired ability to repair the integrity of the barrier and re-establishing tissue homeostasis after the insult. The barrier was repaired much more slowly and even 5–6 days after insult, when WT skin was fully repaired, the mutants still showed a signiﬁcant TEWL (Fig. 3e,f). Notably, this barrier-repair defect could be rectiﬁed by adding topically rIL-13 after the insult—both in mice lacking canonical IELs and in mice lacking IL-13 (Fig. 3g,h). Indeed, even topical application of IL-13 cytokine to WT mice after removal of the stratum corneum (Fig. 3c,d). Moreover, when the skin of mice lacking canonical IELs or IL-13 was challenged by removing the stratum corneum by tape-stripping, an increased degree of water loss was observed, suggesting a worse barrier function and a poorer tolerance to damage (Fig. 3e,f, inserts). More strikingly, mice deﬁcient in IELs or IL-13 showed a signiﬁcantly impaired ability to repair the integrity of the barrier and re-establishing tissue homeostasis after the insult. The barrier was repaired much more slowly and even 5–6 days after insult, when WT skin was fully repaired, the mutants still showed a signiﬁcant TEWL (Fig. 3e,f). Notably, this barrier-repair defect could be rectiﬁed by adding topically rIL-13 after the insult—both in mice lacking canonical IELs and in mice lacking IL-13 (Fig. 3g,h). Indeed, even topical application of IL-13 cytokine to WT mice after removal of the stratum corneum sped-up the recovery of an intact barrier (Supplementary Fig. 6A,B). However, for deeper skin insults beyond the epithelium, IL-13 was redundant in the repair of full skin thickness wounds (Supplementary Fig. 7), although closure of full-thickness skin wounds are signiﬁcantly delayed in gd T-cell-deﬁcient mice19,20. Thus, skin IELs and their IL-13 production are vital in maintaining a healthy epithelium and play an important role in restoring epithelial tissue integrity following insult. IL-13 protects against cutaneous epithelial carcinogenesis. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms12080 (c–e) Primary neonatal KCs cultured in vitro with/without 20 ng ml 1 rIL-13 were assessed for expression of (c) TSLP, (d) IL-1a and (e) TNFa (n ¼ 6). UT, untreated. (a–e) data from WT FVB mice. (f–h) WTand IL-13 / mice were treated topically in vivo with DMBA once and freshly isolated epidermis analysed by qRT–PCR 72 h later for expression of the genes (f) TSLP, (g) IL-33 and (h) caspase-3. (i,j) Fully reconstituted BM chimera mice were treated with a single skin exposure of DMBA (8 weeks after BM transplant) and isolated epidermis analysed for (i) TSLP and (j) IL-33 expression at the indicated time points (n ¼ 4 per time point). IL-13 / -WT indicates that the donor is IL-13 / and the host WT (IL-13-sufﬁcient IELs); WT-IL-13 / indicates that the donor is WTand the host IL-13 / (IL-13-deﬁcient IELs). Data in c–j were done by qRT–PCR and are expressed as mean±1 s.e.m. relative to the control gene cyclophylin. WT mice are shown in black bars and IL-13 / mice in orange bars. (f–j) Data from mice on BALB/c background. Statistical signiﬁcance of difference between experimental groups was determined using Student’s t-test for unpaired data with Po0.05, Po0.01, Po0.001 and *Po0.0001. IEL-derived IL-13 is central for a canonical KC stress response, demonstrating the importance of an integrated local tissue response to acute environmental challenges. and had signiﬁcant aberrant expression of the NKG2D ligand Rae-1 when canonical IELs were absent. This was evident also in total IL-13-deﬁcient mice and even in mice where IL-13 was absent only in the IELs, while ECs in mice with IL-13-sufﬁcent IELs had no or low expression of Rae-1 in spite of lacking IL-13 in the hematopoietic system (Fig. 3a,b). In addition, quantiﬁcation of TEWL as a measure of skin integrity showed that even the resting skin had a small but signiﬁcantly higher degree of water loss in the absence of canonical IELs or IL-13 IEL and IL-13 sustain tissue health and restore integrity. To determine the relevance of IELs and the role of IL-13 on the health status of the epithelial tissue, we ﬁrst looked at the resting steady-state skin. We found that the ECs appeared more stressed NATURE COMMUNICATIONS \| 7:12080 \| DOI: 10.1038/ncomms12080 \| www.nature.com/naturecommunications 4 4 NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms12080 Rae-1 expression in the epide tissue health and restore integrity after insult. Rae-1 expression in the epidermis of naive (a) FVB WT (n ¼ 5), Figure 3 \| IELs and IL-13 maintain tissue health and restore integrity after insult. Rae-1 expression in the epidermis of naive (a) FVB WT (n ¼ 5), FVB Tcrd / (n ¼ 5) and (B) WT BALB/c /IL-13 / BM chimeras (n ¼ 4 per group) was analysed by qRT–PCR. Data are expressed as mean±1 s.e.m. relative to the control gene cyclophilin. In b, IL-13 / donor to WT have IL-13-sufﬁcient IELs; WT donor to IL-13 / host have IL-13-deﬁcient IELs. (c–h) TEWL was analysed as a measure of barrier integrity using a tewameter probe assessing water evaporation rate and reported as g hm 2 (where g ¼ water loss in grams, h ¼ time in hours, m2 ¼ metres squared). TEWL was measured in naive (c) FVB WTand Tcrd / mice (n ¼ 20 per group) as well as (d) BALB/c WTand IL-13 / mice (n ¼ 25–27 per group). (e,f) The dorsal ear skin was abraded by tape-stripping (6 ) and TEWL measured just after tape-stripping and at indicated time points the following days in (e) FVB WT and Tcrd / and (f) BALB/c WT and IL-13 / mice (n ¼ 10 in all groups). The data are expressed as % of the max TEWL measured just after tape-stripping. Max TEWL as measured just after tape-stripping is shown for all groups in the insert graphs. (e,f) were repeated twice with similar results. Black lines show WTgroups, red line Tcrd / and orange line IL-13 / mice. (g,h) The dorsal ear skin was abraded by tape-stripping (6 ) and TEWL measured as before but (g) Tcrd / and (h) IL-13 / mice were treated topically with 50 ng rIL-13 just after tape-stripping and again after 24 h (n ¼ 10 in all groups). (g) Red line shows Tcrd / mice þ rIL-13 and (h) orange line shows IL-13 / mice þ rIL-13, WT mice treated with the vehicle, PBS, is shown in black lines. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms12080 Rae-1 expression in the epidermis of naive (a) FVB WT (n ¼ 5), FVB Tcrd / (n ¼ 5) and (B) WT BALB/c /IL-13 / BM chimeras (n ¼ 4 per group) was analysed by qRT–PCR. Data are expressed as mean±1 s.e.m. relative to the control gene cyclophilin. In b, IL-13 / donor to WT have IL-13-sufﬁcient IELs; WT donor to IL-13 / host have IL-13-deﬁcient IELs. (c–h) TEWL was analysed as a measure of barrier integrity using a tewameter probe assessing water evaporation rate and reported as g hm 2 (where g ¼ water loss in grams, h ¼ time in hours, m2 ¼ metres squared). TEWL was measured in naive (c) FVB WTand Tcrd / mice (n ¼ 20 per group) as well as (d) BALB/c WTand IL-13 / mice (n ¼ 25–27 per group). (e,f) The dorsal ear skin was abraded by tape-stripping (6 ) and TEWL measured just after tape-stripping and at indicated time points the following days in (e) FVB WT and Tcrd / and (f) BALB/c WT and IL-13 / mice (n ¼ 10 in all groups). The data are expressed as % of the max TEWL measured just after tape-stripping. Max TEWL as measured just after tape-stripping is shown for all groups in the insert graphs. (e,f) were repeated twice with similar results. Black lines show WTgroups, red line Tcrd / and orange line IL-13 / mice. (g,h) The dorsal ear skin was abraded by tape-stripping (6 ) and TEWL measured as before but (g) Tcrd / and (h) IL-13 / mice were treated topically with 50 ng rIL-13 just after tape-stripping and again after 24 h (n ¼ 10 in all groups). (g) Red line shows Tcrd / mice þ rIL-13 and (h) orange line shows IL-13 / mice þ rIL-13, WT mice treated with the vehicle, PBS, is shown in black lines. Statistical signiﬁcance in a–d and inset graphs in e–f were determined using Student’s t-test for unpaired data with Po0.01, *Po0.001 and Po0.0001. In e–h, statistical signiﬁcance of difference between experimental groups was assessed over time using linear regression. NS, not signiﬁcant. Figure 3 \| IELs and IL-13 maintain tissue health and restore integrity after insult. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms12080 The data are expressed as % of the max TEWL measured just after tape-stripping. Max TEWL as measured just after tape-stripping is shown for all groups in the insert graphs. (e,f) were repeated twice with similar results. Black lines show WTgroups, red line Tcrd / and orange line IL-13 / mice. (g,h) The dorsal ear skin was abraded by tape-stripping (6 ) and TEWL measured as before but (g) Tcrd / and (h) IL-13 / mice were treated topically with 50 ng rIL-13 just after tape-stripping and again after 24 h (n ¼ 10 in all groups). (g) Red line shows Tcrd / mice þ rIL-13 and (h) orange line shows IL-13 / mice þ rIL-13, WT mice treated with the vehicle, PBS, is shown in black lines. Statistical signiﬁcance in a–d and inset graphs in e–f were determined using Student’s t-test for unpaired data with Po0.01, *Po0.001 and Po0.0001. In e–h, statistical signiﬁcance of difference between experimental groups was assessed over time using linear regression. NS, not signiﬁcant. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms12080 ARTICLE WT Tcrd–/– 0.00 0.01 0.02 0.03 Relative expression of Rae-1 0.00 0.05 0.10 0.15 Relative expression of Rae-1 b a * ** WT WT IL-13–/– IL-13–/– IL-13–/– WT WT IL-13–/– Donor: Host: WT Tcrd–/– 0 2 4 6 8 10 TEWL (g hm–2) c ** WT Tcrd–/– 0.00 0.01 0.02 0.03 Relative expression of Rae-1 a TEWL (g hm–2) WT IL-13–/– 0 2 4 6 8 10 d d WT TEWL (g hm–2) 0 20 40 60 80 100 120 0 20 40 60 80 100 Time (h) % of max TEWL IL-13–/– 60 70 80 90 100 110 f P=0.0480 3–/– 0 20 40 60 80 100 0 20 40 60 80 100 Time (h) WT Tcrd–/– 50 60 70 80 90 100 TEWL (g hm–2) % of max TEWL e *** P=0.0011 f e Time (h) % of max TEWL 0 20 40 60 80 100 0 20 40 60 80 100 Time (h) h P = NS Time (h) 0 20 40 60 80 100 0 20 40 60 80 100 % of max TEWL Time (h) g P=NS Figure 3 \| IELs and IL-13 maintain tissue health and restore integrity after insult. NATURE COMMUNICATIONS \| 7:12080 \| DOI: 10.1038/ncomms12080 \| www.nature.com/naturecommunications ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms12080 type-2 immunity9,12 and local IL-13 responses (Fig. 1). Hence, we wanted to examine the role of IL-13 in epithelial carcinogenesis. We assessed skin tumour formation in IL-13-deﬁcient mice using two different models of spontaneous epithelial carcinogenesis. First, we evoked ‘DMBA complete’ carcinogenesis by exposing the back skin once weekly to the carcinogen DMBA. This does not cause inﬂammation in the tissue, but the ensuing accumulation of mutations in the ECs causes the outgrowth of tumours. Mice lacking IL-13 were signiﬁcantly more susceptible to DMBA-induced carcinogenesis and developed both more and bigger tumours than WT mice (Fig. 4a,b). Mice deﬁcient in canonical IELs were also highly susceptible to DMBA complete carcinogenesis, developing tumours earlier, as well as signiﬁcantly more and bigger tumours than their WT counterparts (Fig. 4c,d). Next, we examined the commonly used model of DMBA–TPA carcinogenesis, where a single subcarcinogenic dose of DMBA, which causes EC mutations primarily in hras (initiation), is followed by repeated applications of the inﬂammatory reagent TPA (promotion), which allows for the few mutated clones to expand and form tumours. gd T-cell-deﬁcient and Vg5Vd1 þ IEL-deﬁcient mice are more susceptible to this form of two-stage chemical cutaneous carcinogenesis9,15. Consistent with this, mice lacking IL-13 were also more susceptible to this inﬂammation-driven carcinogenesis and developed signiﬁcantly more and bigger tumours than WT controls (Fig. 4e,f). This is speciﬁc to IL-13 as mice lacking IL-4 were signiﬁcantly less susceptible to tumour development following DMBA–TPA, suggesting that IL-4 may drive inﬂammation in this model, whereas IL-13 works differently (Fig. 4g,h). A constraint on this interpretation is the caveat that the IL-13 and IL-4 mutant mice used in this study were on a different background strain. There are well-known differences in susceptibility to DMBA– TPA carcinogenesis across different background strains21, making direct comparison between mutants challenging. All experiments on mutant strains were internally controlled with the appropriate background strain. Furthermore, using BM chimeric mice, we were additionally able to show that mice lacking IL-13 only in the IELs (WT-IL-13 / ) were as susceptible to DMBA carcinogenesis as mice completely lacking IL-13 and both mutants developed more tumours than WT (Fig. 4i). ARTICLE KCs move from the basal proliferative layer up through the spinal and granular layers undergoing differentiation 5 10 15 20 0 10 20 30 40 Weeks post initiation WT IL-13–/– P < 0.0001 f Tumour area / mouse (mm2) Weeks post initiation Weeks post initiation 5 10 15 20 0 2 4 6 8 Weeks post initiation Tumours / mouse WT IL-13–/– P<0.0001 e f Tumour area / mouse (mm2) f e e f Tumour area / mouse 2 Figure 4 \| IELs and IL-13 protect against cutaneous epithelial carcinogenesis. (a–d) The shaved backs of mice were treated once weekly with 200 nmol DMBA to evoke ‘complete DMBA’ carcinogenesis. Animals were scored, tumours counted and measured once weekly. Data are expressed as average number of tumours per (a) BALB/c WT and IL-13 / mouse, and (c) FVB WT and Tcrd / mouse, and average tumour size per (b) BALB/c WTand IL-13 / mouse, and (d) FVB WTand Tcrd / mouse (n ¼ 10–14 per group). (e–h) The shaved backs of mice were treated with a single subcarcinogenic dose of DMBA followed by twice weekly application of TPA to promote chronic skin inﬂammation and outgrowth of tumours. Mice were scored weekly and the data expressed as (e,g) average tumour number or (f,h) tumour size per (e,f) BALB/c WT or IL-13 / mouse (n ¼ 12 per group) or (g,h) FVB WT or IL-4 / mouse (n ¼ 10 per group). Statistical signiﬁcance between groups was assessed over time using linear regression. (i) BALB/c WT/IL-13 / BM chimera mice were generated as described and 8 weeks later the mice were subjected to ‘DMBA complete’ carcinogenesis. Data show the average number of tumours per mouse at the end of the experiment, week 17. WT donor to IL-13 / host have IL-13-deﬁcient IELs (n ¼ 8 per group). Statistical signiﬁcance of difference between experimental groups was determined using Student’s t-test for unpaired data with Po0.05 and Po0.01. NS, not signiﬁcant. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms12080 We have shown that the same IEL that is host-protective against inﬂammation-driven carcinogenesis is also a strong driver of NATURE COMMUNICATIONS \| 7:12080 \| DOI: 10.1038/ncomms12080 \| www.nature.com/naturecommunications 5 5 NATURE COMMUNICATIONS \| 7:12080 \| DOI: 10.1038/ncomms12080 \| www.nature.com/naturecommunications ARTICLE The increased susceptibility of IL-13 / mice to DMBA carcinogenesis was not due to an increased absorption of the carcinogen through the barrier-disrupted skin or an increased metabolism of DMBA due to differences in LCs or other skin DC populations (Supplementary Fig. 8), as the level of double-stranded DNA breaks in ECs caused by the initial DMBA exposure was similar between WT and IL-13 / mice (Supplementary Fig. 9). The tumour-protective effect of IL-13 additionally appears to be restricted to carcinogenesis at the epithelial skin barrier, as mice lacking IL-13 were not more susceptible to subcutaneous tumour growth (Supplementary Fig. 10). Together, this demonstrates that IL-13 is strongly protective against cutaneous epithelial carcinogenesis. ARTICLE 5 10 15 0 50 100 5 10 15 0 2 4 6 8 Weeks post initiation Tumours / mouse WT IL-13–/– P=0.0012 5 10 15 0 2 4 6 8 10 Weeks post initiation Tumours / mouse WT Tcrd–/– P<0.0001 Tcrd–/– P < 0.0001 5 10 15 0 10 20 30 40 Weeks post initiation P=0.0027 5 10 15 20 0 2 4 6 8 Weeks post initiation Tumours / mouse WT IL-13–/– P<0.0001 5 10 15 20 0 10 20 30 40 Weeks post initiation WT WT IL-13–/– WT IL-13–/– P < 0.0001 0 5 10 15 Tumours / mouse week 17 WT WT WT IL-13–/– IL-13–/– IL-13–/– Donor: Host: * * NS a b c d e f 0 5 10 15 0 10 20 30 40 Weeks post initiation Tumours / mouse WT IL-4–/– 0 5 10 15 0 50 100 150 200 Weeks post initiation Tumour area / mouse (mm2) Tumour area / mouse (mm2) Tumour area / mouse (mm2) Tumour area / mouse (mm2) WT IL-4–/– P < 0.0001 P < 0.0001 g h i Weeks post initiation 6 NATURE COMMUN b Tumour area / mouse ( 2) 5 10 15 0 2 4 6 8 Weeks post initiation Tumours / mouse WT IL-13–/– P=0.0012 a 5 10 15 0 10 20 30 40 Weeks post initiation P=0.0027 WT IL-13–/– b Tumour area / mouse (mm2) a p 5 10 15 0 2 4 6 8 10 Weeks post initiation Tumours / mouse WT Tcrd–/– P<0.0001 c 5 10 15 0 50 100 Tcrd–/– P < 0.0001 WT d Tumour area / mouse (mm2) Weeks post initiation d Tumour area / mouse (mm2) d c Tumour area / mouse ( 2) 10 15 Weeks post initiation IL-13 promotes EC maturation and transit through epidermis. To explore how IL-13 promotes tissue integrity and protects against carcinogenesis, we studied its effect on epidermal structure and maintenance. The skin epithelium is under constant renewal. ARTICLE Both IELs, resident in the epidermis, and ILC2s, resident in the dermis, are constitutively positive for IL-13. ILC2s are increasingly acknowledged to be regulating cutaneous immune response26,27; however, they appear redundant in supporting acute EC responses in the epidermis. ILCs are ablated by radiation and re-populated by BM cells (data not shown and refs 26,28), whereas IELs are radioresistant, so by generating BM chimeras we could reveal the important role of IEL-derived IL-13 for homeostatic responses in the epithelium. Overall, our study demonstrates that constitutive presence of IL-13 in the epidermis promotes barrier integrity and protects against carcinogenesis. g g The regulation and function of type-2 immunity remain somewhat enigmatic. What is known so far of the physiological role of type-2 responses is that their host-protection properties converge in different forms of barrier defences29. Much of type-2 immunity appears dedicated to tissue repair and promoting tolerance to damage. Our data on IL-13 in the epidermis ﬁts with this idea and is consistent with the previous data from gd T-cell-deﬁcient mice showing defects in the integrity of the epidermal barrier as well as a predisposition to develop spontaneous dermatitis30,31. A similar protective role for IL-13Ra1 signalling in ECs during lung injury and homeostasis has recently been reported32. This role for IL-13 in supporting ‘EC health’ contrasts with the prevailing paradigm in AD, where the over-production of IL-13 is thought partly responsible for the abnormal epithelial barrier phenotype33,34. Type-2 cytokines are clearly upregulated in the barrier-disrupted skin in mice and in humans with AD35–37, but whether this is the cause or effect of skin disease has long been debated. In AD, most of the type-2 cytokines comes from the inﬂammatory inﬁltrate and it may well be that the ‘over-production’ of IL-13 in this setting is too much/at the wrong time/from the wrong cells and is indeed detrimental for epidermal stability as studies from in vitro grown KC suggest34,38. However, as shown here, during acute insults, the homeostatic IL-13 response in vivo is clearly important for pushing basal KCs upwards and thereby aids the replenishing of the upper mature epidermis (Figs 5 and 3). This role for IL-13 as an ‘epithelial escalator’ has previously been demonstrated in the gut epithelium, where it is thought that this is why IL-13 is essential for gut parasite expulsion39. ARTICLE 5e,f), suggesting that IL-13 promotes upward-moving KC maturation. This was supported by IL-13-induced expression of products associated with terminal differentiation such as caspase-3 both in vivo and in vitro (Figs 2h and 5g)22. To explore this further, we studied the rate of KC transit through the epidermis in a bromodeoxyuridine (BrdU) pulse-chase experiment. BrdU þ cells were counted in the basal epidermal layer along the basement membrane and compared with the number of BrdU þ cells in the granular layers; the role of IL-13 in this KC turnover and migration was evaluated at steady state and following topical exposure to TPA. In steady-state resting tissue, there were only few proliferating cells, these were nearly all in the basal layer—and there was no obvious difference between WT and IL-13-deﬁcient mice (Fig. 5h). Following topical TPA application, many more KCs were proliferating and approximately half of these had moved into the granular layer in WT. However, in mice lacking IL-13, although more cells were proliferating than in the resting skin, these were nearly all in the basal layer and only very few had moved into the granular layers (Fig. 5i,k). This was particularly evident if the ratio of granular to basal BrdU þ cells was compared (Fig. 5j), illustrating that the KCs were migrating at a faster rate in IL-13-sufﬁcient mice. As a result, the epidermis was also thinner in mice lacking IL-13 (Fig. 5k), consistent with previous reports showing that transgenic overexpression of IL-13 in basal KCs induces thickening of the epidermal layer23. Overall, the thickening of the total ear skin was reduced following topical TPA in IL-13-deﬁcent mice (Fig. 5l). Together, this shows that the rate of EC movement from the basal layer is under control of IL-13 and hence IL-13 contributes to the upwards maturation of skin ECs and epithelial renewal. y We show here that skin-resident gd TCR þ IELs are potent producers of IL-13, following a variety of environmental stressors and this greatly affects EC function with pleiotropic consequences for tissue health. Human epidermal gd T cells have likewise been shown to be high producers of type-2 cytokines—and particularly IL-13 (ref. 25). While much has been published on the effects of excessive expression of IL-4/IL-13 on the skin barrier in inﬂammatory skin diseases such as atopic dermatitis (AD), the potential role of constitutive expression of IL-13 has hitherto been less well understood. ARTICLE Indeed, a recent study expands on this, and supports our described role of IL-13 in controlling EC fate decisions, by showing how IL-13 from intestinal ILCs regulate the cellular composition of the gut epithelium by signalling to uncommitted intestinal ECs40. This effect on EC transit time may possibly contribute to carcinogenesis susceptibility. In support of this, mice lacking canonical epidermal IELs had a higher level of mutant hras (A-T transversion within hras codon 61) in the epidermis following DMBA exposure but before clinical signs of disease, suggesting that the mutated cells accumulate in the tissue (Supplementary Fig. 12). ARTICLE ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms12080 and maturation before entering terminal differentiation and programmed cell death to form the corniﬁed stratum corneum. To maintain tissue integrity and stability KCs express a multitude of structural proteins and complexes of scaffolding and adhesive proteins, which are tightly regulated during KC differentiation. In line with the barrier-repair defect that we found in the absence of IL-13 (Fig. 3), we also consistently found a reduced expression of tight junction proteins and structural components of the upper epidermis in IL-13-deﬁcient mice when challenged in vivo to repair the barrier following tape-stripping (Fig. 5a–d). While BALB/c WT mice upregulated the expression of claudin-1, occludin and transglutaminase 1 at 24–48 h after the insult, in accordance with morphological tissue repair, this response was diminished in the absence of IL-13. FVB mice lacking epidermal IELs also showed diminished expression of claudin-1 and trans- glutaminase 1 after insult, but the kinetics of the response was slightly different on the FVB strain background (Supplementary Fig. 11). In addition, adding rIL-13 to primary KCs in vitro strongly downregulated genes associated with basal KCs such as K5 and K15 (Fig. 5e,f), suggesting that IL-13 promotes upward-moving KC maturation. This was supported by IL-13-induced expression of products associated with terminal differentiation such as caspase-3 both in vivo and in vitro (Figs 2h and 5g)22. To explore this further, we studied the rate of KC transit through the epidermis in a bromodeoxyuridine (BrdU) pulse-chase experiment. BrdU þ cells were counted in the basal epidermal layer along the basement membrane and compared with the number of BrdU þ cells in the granular layers; the role of IL-13 in this KC turnover and migration was evaluated at steady state and following topical exposure to TPA. In steady-state resting tissue, there were only few proliferating cells, these were nearly all in the basal layer—and there was no obvious difference between WT and IL-13-deﬁcient mice (Fig. 5h). Following topical TPA application, many more KCs were proliferating and approximately half of these had moved into the granular layer in WT. However, in mice lacking IL-13, although more cells were proliferating than in the resting skin, these were nearly all in the basal layer and only very few had moved into the granular layers (Fig. 5i,k). This was particularly evident if the ratio of granular to basal BrdU þ cells was compared (Fig. ARTICLE 5j), illustrating that the KCs were migrating at a faster rate in IL-13-sufﬁcient mice. As a result, the epidermis was also thinner in mice lacking IL-13 (Fig. 5k), consistent with previous reports showing that transgenic overexpression of IL-13 in basal KCs induces thickening of the epidermal layer23. Overall, the thickening of the total ear skin was reduced following topical TPA in IL-13-deﬁcent mice (Fig. 5l). Together, this shows that the rate of EC movement from the basal layer is under control of IL-13 and hence IL-13 contributes to the upwards maturation of skin ECs and epithelial renewal. This effect on EC transit time may possibly contribute to carcinogenesis susceptibility In support of this mice lacking T cells can be direct afferent sensors of EC dysregulation9,12. This study provides insight into one mechanism, whereby gd TCR þ IELs contribute to immune surveillance through direct action on neighbouring ECs, promoting tissue repair and protection against tumour formation. A general picture of an important role for gd T cells, which are highly tissue-tropic, in cancer immune surveillance in both mouse and human is emerging. A recent large meta-analysis, integrating tumour gene expression with survival data, revealed the presence of intra-tumoral gd T cells as the most signiﬁcant favourable prognostic population of all leukocytes across human cancers24. and maturation before entering terminal differentiation and programmed cell death to form the corniﬁed stratum corneum. To maintain tissue integrity and stability KCs express a multitude of structural proteins and complexes of scaffolding and adhesive proteins, which are tightly regulated during KC differentiation. In line with the barrier-repair defect that we found in the absence of IL-13 (Fig. 3), we also consistently found a reduced expression of tight junction proteins and structural components of the upper epidermis in IL-13-deﬁcient mice when challenged in vivo to repair the barrier following tape-stripping (Fig. 5a–d). While BALB/c WT mice upregulated the expression of claudin-1, occludin and transglutaminase 1 at 24–48 h after the insult, in accordance with morphological tissue repair, this response was diminished in the absence of IL-13. FVB mice lacking epidermal IELs also showed diminished expression of claudin-1 and trans- glutaminase 1 after insult, but the kinetics of the response was slightly different on the FVB strain background (Supplementary Fig. 11). In addition, adding rIL-13 to primary KCs in vitro strongly downregulated genes associated with basal KCs such as K5 and K15 (Fig. NATURE COMMUNICATIONS \| 7:12080 \| DOI: 10.1038/ncomms12080 \| www.nature.com/naturecommunications ARTICLE Weeks post initiation Weeks post initiation 0 5 10 15 0 10 20 30 40 Weeks post initiation Tumours / mouse WT IL-4–/– 0 5 10 15 0 50 100 150 200 Weeks post initiation Tumour area / mouse (mm2) WT IL-4–/– P < 0.0001 P < 0.0001 g h h g 0 5 10 15 Tumours / mouse week 17 WT WT WT IL-13–/– IL-13–/– IL-13–/– Donor: Host: ** * NS i 0 5 10 15 Tumours / mouse week 17 WT WT WT IL-13–/– IL-13–/– IL-13–/– Donor: Host: ** * NS i 6 NATURE COMMUNICATIONS \| 7:12080 \| DOI: 10.1038/ncomms12080 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms12080 of BrdU+ cells 0 50 100 150 200 0.0 0.2 0.4 0.6 0.8 1.0 Ratio of BrdU+ cells Ratio of granular to basal BrdU+ cells * h i j UT + rIL-13 UT + rIL-13 UT + rIL-13 0.00 0.01 0.02 0.03 0.04 Relative expression of K5 0.000 0.005 0.010 0.015 Relative expression of K15 * *** e f 0.000 0.001 0.002 0.003 Relative expression of Casp 3 * g WT IL-13–/– Time (h) 0 20 40 60 80 Ear thickness (mm) 0 24 72 0 24 72 * k l BrdU NATURE COMMUNICATIONS \| 7:12080 \| DOI: 10.1038/ncomms12080 \| www.nature.com/naturecommunications 0 2 6 18 24 48 0.000 0.005 0.010 Time (h) Relative expression of claudin 1 WT IL-13–/– 0 2 6 18 24 48 0.0000 0.0002 0.0004 0.0006 Time (h) Relative expression of occludin a b 0 2 6 18 24 48 0.0000 0.0002 0.0004 0.0006 Time (h) Relative expression of occludin ** b 0 2 6 18 24 48 0.000 0.005 0.010 Time (h) Relative expression of claudin 1 WT IL-13–/– ** a b a Time (h) Time (h) WT IL-13–/– Time (h) Time (h) 0 2 6 18 24 48 0.0000 0.0002 0.0004 0.0006 Relative expression of Tgm1 0 2 6 18 24 48 0.000 0.005 0.010 0.015 0.020 Relative expression of Cdsn * * ** ** c d UT + rIL-13 UT + rIL-13 UT + rIL-13 0.00 0.01 0.02 0.03 0.04 Relative expression of K5 0.000 0.005 0.010 0.015 Relative expression of K15 * *** e f 0.000 0.001 0.002 0.003 Relative expression of Casp 3 * g Time (h) Time (h) WT IL-13–/– Time (h) Time (h) 0 2 6 18 24 48 0.0000 0.0002 0.0004 0.0006 Relative expression of Tgm1 0 2 6 18 24 48 0.000 0.005 0.010 0.015 0.020 Relative expression of Cdsn * * ** ** c d Time (h) WT IL-13–/– Time (h) 0 2 6 18 24 48 0.0000 0.0002 0.0004 0.0006 Relative expression of Tgm1 * * c Time (h) Time (h) 0 2 6 18 24 48 0.000 0.005 0.010 0.015 0.020 Relative expression of Cdsn ** ** d d d c ( ) ( ) UT + rIL-13 UT + rIL-13 UT + rIL-13 0.00 0.01 0.02 0.03 0.04 Relative expression of K5 0.000 0.005 0.010 0.015 Relative expression of K15 * *** e f 0.000 0.001 0.002 0.003 Relative expression of Casp 3 * g UT + rIL-13 0.00 0.01 0.02 0.03 0.04 Relative expression of K5 * e UT + rIL-13 UT + rIL-13 0.000 0.005 0.010 0.015 Relative expression of K15 *** f 0.000 0.001 0.002 0.003 Relative expression of Casp 3 * g f g e Basal Granular Basal Granular Basal Granular Basal Granular 0 2 4 6 8 No. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms12080 NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms12080 In terms of early cancer immune surveillance, the role of type-2 immunity has been little explored; the focus having been ﬁrmly on type-1 immunity and cytotoxic mechanisms, both of which have strong experimental support for playing a role in extrinsic tumour suppression. Nonetheless, the repair functions of type-2 immune surveillance are clearly important in protection against carcinogenesis, as illustrated by the link between wounding and tumour development41. Tumours can develop at the site of chronic skin wounds42,43 and patients with epidermolysis bullosa, who have chronic skin damage, are at increased risk of developing squamous cell carcinoma44,45. A diminished capacity to repair a damaged barrier can thus predispose to the development of cancer. Our data support this link, as deﬁciency in IL-13 resulted in both a barrier defect with reduced capacity to restore tissue p pp p y g ssion. Nonetheless, the repair functions of type-2 llance are clearly important in protection against as illustrated by the link between wounding and q p y p damaged barrier can thus predispose to the development of cancer. Our data support this link, as deﬁciency in IL-13 resulted in both a barrier defect with reduced capacity to restore tissue 0 2 6 18 24 48 0.000 0.005 0.010 Time (h) Relative expression of claudin 1 WT IL-13–/– 0 2 6 18 24 48 0.0000 0.0002 0.0004 0.0006 Time (h) Relative expression of occludin ** a b WT IL-13–/– Time (h) Time (h) 0 2 6 18 24 48 0.0000 0.0002 0.0004 0.0006 Relative expression of Tgm1 0 2 6 18 24 48 0.000 0.005 0.010 0.015 0.020 Relative expression of Cdsn * * ** c d Basal Granular Basal Granular Basal Granular Basal Granular 0 2 4 6 8 No. of BrdU+ cells No. Discussion Immune surveillance can function by many (non-exclusive) mechanisms; it can recognize and remove damaged cells, remove or neutralize potential harmful environmental substances, facilitate re-establishment of homeostasis by tissue repair and dampen detrimental inﬂammation. Tissue-resident immunocytes are in a unique position to carry out a continued maintenance function such as immune stress surveillance and tissue-resident 7 7 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms12080 of BrdU+ cells No. of BrdU+ cells 0 50 100 150 200 0.0 0.2 0.4 0.6 0.8 1.0 Ratio of BrdU+ cells Ratio of granular to basal BrdU+ cells * h i j Basal Granular Basal Granular No. of BrdU+ cells 0 50 100 150 200 0.0 0.2 0.4 0.6 0.8 1.0 Ratio of BrdU+ cells Ratio of granular to basal BrdU+ cells *** i j h i WT IL-13–/– Time (h) 0 20 40 60 80 Ear thickness (mm) 0 24 72 0 24 72 * k l BrdU Time (h) 0 20 40 60 80 Ear thickness (mm) 0 24 72 0 24 72 * l WT IL-13–/– k BrdU k NATURE COMMUNICATIONS \| 7:12080 \| DOI: 10.1038/ncomms12080 \| www.nature.com/naturecommunications 8 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms12080 to play distinct roles in asthma55. Indeed, the spatial and temporal expression of these divergent cytokines may also be very important in AD and further mechanistic studies are warranted to understand the role of type-2 immunity in regulating skin barrier function. integrity on insult and a signiﬁcantly higher susceptibility to cutaneous carcinogenesis (Figs 3 and 4). A protective role for IL-13 in inﬂammation-driven skin carcinogenesis has previously been reported46 and ‘atopic’ mice have likewise been shown to be protected36. Cipolat et al. showed that mice with a genetic barrier defect had an exaggerated type-2 response, including IL-13 and TSLP, and were protected against DMBA–TPA carcinogenesis. The ‘atopic’ phenotype could be inhibited by blocking TSLP or NKG2D36, suggesting that LSS responses are induced by barrier disruption and protect against carcinogenesis also in this model. A homeostatic IL-13 response thus ensures normal tissue integrity (Fig. 3), but enhanced responses can induce hyperplasia and thickening of the epidermis (Fig. 5), which may primarily be driven by TSLP47. Acute hyperplasia results in improved resistance to damage and damaging substances at the body barrier and may therefore be another protective mechanism against carcinogenesis. The data presented here, showing a protective role for IL-13 in different models of epithelial carcinogenesis, provides new perspective to further studies on the links between atopy and cancer. The fact that IELs and IL-13 protect against epithelial carcinogenesis, in both mutation- and inﬂammation-driven models, suggest that, in contrast to IL-4, this IEL–EC axis of communication plays a central regulatory role in addition to suppressing inﬂammation. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms12080 This may be due to the role of IELs and IL-13 in controlling the rate of EC movement through the epidermis. IL-13 accelerates EC turnover and transit towards the upper epidermis. An increased ‘transit time’ of skin ECs from the basal proliferative layer to the outer cell layers have previously been shown to be tumour suppressive in skin carcinogenesis56. Similarly, mice lacking the major structural protein keratin 10 in the upper epidermis have a strongly accelerated EC turnover and are signiﬁcantly protected against skin carcinogenesis, suggesting an increased elimination of initiated KCs57. Conversely, skin cells resistant to terminal differentiation have since long been associated with initiation of carcinogenesis58. py The association between defective tissue repair and cancer development may not only pertain to the lack of repair per se but also to the detrimental inﬂammation caused by chronic wounds. There is a close link between chronic inﬂammation and cancer, and once a malignant cell has escaped the early phase of immune surveillance, inﬂammation can exert prominent pro-carcinogenic effects48. An important component of early tumour surveillance could hence be the release of anti-inﬂammatory products in the tissue. IL-13 can have direct anti-inﬂammatory effects in the epithelial tissue as shown in the intestinal epithelium49, but recently a lot of focus has been on the more newly discovered EC cytokines—IL-33, TSLP and IL-25. Skin ECs promptly produce particularly IL-33 and TSLP upon stress, as when exposed to the carcinogen DMBA. Interestingly, this canonical EC stress response is under control of IEL-derived IL-13 (Fig. 2f–j). TSLP has been implicated in the development and progression of allergic diseases, in both human and mouse, in many recent studies and is thought to be a central regulator of atopy in several epithelial tissues50. TSLP is, however, also emerging as a potent tumour suppressor51. In the skin, TSLP is strongly protective against carcinogenesis52,53 and this is thought to be due to EC-derived TSLP perturbing the balance of inﬂammation in the tissue53. Clearly, not all type-2 immunity is anti-inﬂammatory. In this study, we show an interesting divergent effect of the type-2 cytokines IL-4 and IL- 13 in inﬂammation-driven carcinogenesis. While IL-13 is protective, IL-4 clearly promotes tumour growth in this model (Fig. 4) and is mainly produced by the inﬂammatory inﬁltrate. Methods Mi IL 13 Mice. IL-13-egfp reporter mice were generated as described61 and bred to homozygosity for use as IL-13 / . IL-13-egfp mice were on BALB/c strain background. Tcrd / mice62 and IL-4 / were backcrossed onto FVB/N background 410 generations. BALB/c and FVB/N WT mice were purchased from Charles River and used as controls. For experiments requiring neonatal KCs, neonatal mice were bred in-house. Mice were bred and maintained in individually ventilated cages under speciﬁc pathogen-free conditions; with food and water provided ad libitum. Age-matched, female mice were used for all experiments at Z7 weeks of age. All studies were approved by Imperial College AWERB (Animal Welfare and Ethical Review Body) and the UK Home Ofﬁce for Laboratory Animal Care regulations. Experiments involving cancer studies strictly adhered to the guidelines set out by the National Cancer Research Institute (NCRI) and Workman et al.63 in ‘Guidelines for the Welfare and Use of Animals in Cancer Research’, and all studies using animals were conducted following the Animal Research: Reporting In Vivo Experiments (ARRIVE) guidelines64. BM chimeras. Mice were sub-lethally irradiated (750 rad) and immediately reconstituted with 5 106 donor BM cells intravenous. Mice were left for 8 weeks, to fully reconstitute the hematopoietic system, and thereafter the reconstitution was Figure 5 \| IL-13 promotes epithelial maturation and transit through the epidermis. Expression analysis of (a) claudin-1, (b) occludin, (c) transglutaminase 1 and (d) corneodesmosin in isolated epidermis from BALB/c WT mice (black bars) and IL-13 / mice (orange bars) at indicated time points after skin abrasion by tape-stripping (nn ¼ 3 per time point). (e–g) Primary neonatal KCs grown in vitro were stimulated with 20 ng ml 1 rIL-13 or left untreated (UT) and analysed for expression of the basal KC genes (e) Keratin 5 and (f) Keratin 15 as well as (g) caspase-3, which is associated with terminal KC differentiation (n ¼ 6). Data in (a–g) were done by qRT–PCR and are expressed as mean±1 s.e.m. relative to the control gene cyclophylin. (h–k) BALB/c WT (black bars) and IL-13 / (orange bars) were treated topically with 10 nmol TPA on the dorsal ear skin or left untreated (n ¼ 3 per group). NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms12080 It is interesting that, although these cytokines share many regulatory elements and have partially shared receptors, their expression pattern is often different54 and they have previously been shown In summary, we demonstrate that IL-13 acts as a molecular bridge between the skin IELs and ECs. This cross-communication regulates EC function and promotes skin homeostasis in a manner that protects against acute challenge and carcinogenesis. LSS is thus part of an early ‘allergic’ type-2 host-defence mechanism aimed at protecting body surface tissues. A similar role for IL-13 in EC protection and homeostasis in the gut39,40 and lung32,59,60 raises the possibility that this may represent a conserved response against noxious environmental substances and damage to ECs—with potential important implications for atopy and cancer. NATURE COMMUNICATIONS \| 7:12080 \| DOI: 10.1038/ncomms12080 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms12080 Cell suspensions were ﬁltered, resuspended in deﬁned KC serum-free medium with supplements (Life) þ 1 antibiotic–antimycotic solution. KCs were seeded at an appropriate cell density onto tissue culture vessels coated with rat-tail-derived-collagen I (Sigma). Culture vessels were washed with PBS 24 h following seeding to remove unattached cells, and provided with fresh medium±20 ng ml 1 recombinant IL-13 (R&D). For extraction of KCs, cell culture vessels were treated with trypsin and KC RNA was subsequently extracted using the RNEasy Mini kit (Qiagen). Tape-stripping and TEWL. The stratum corneum was removed from the ear skin by application and removal of cellophane tape (Scotch) six times per ear. For measurement of TEWL, a tewameter probe (Tewameter TM300; Ck Electronic, Germany) was placed directly onto the ear skin of anaesthetized mice in a temperature- and humidity-controlled facility. Probe readings were analysed and water evaporation rate was reported using the MPA software (MPA software; Ck Electronic, Germany) as g hm 2 (where g ¼ water loss in grams, h ¼ time in hours, m2 ¼ metres squared). For some experiments, mice were treated topically on the ear skin with 50 ng rIL-13 (R&D), or vehicle control, immediately following tape- stripping, and again 24 h later. Epidermal sheet immunoﬂuorescence. Epidermal sheets were ﬁxed in ice-cold acetone at 20 C for 15 min, then rehydrated in PBS and blocked with 2% bovine serum albumin. Sheets were then stained with a ﬂuorescein isothiocyanate- conjugated anti-Vg5 or anti-Langerin antibody and subsequently washed thoroughly in PBS. Sheets were carefully mounted onto slides using anti-fade mounting medium (Dako) and visualised on a Leica SP5 (Leica) confocal microscope. Chemical cutaneous carcinogenesis. The dorsal back area was shaved with a surgical blade and mice rested for 1 week. Applications of chemicals and tumour monitoring were performed as previously described15, 65. Chemicals, in a 100 ml volume, were carefully and slowly applied by pipette to the entire shaved skin area. For ‘DMBA complete’ carcinogenesis, mice were treated once weekly with 200 nmol DMBA. For two-stage ‘DMBA þ TPA’ carcinogenesis, mice were initiated with 200 nmol DMBA followed by twice weekly application of 10 nmol (FVB background) or 20 nmol (BALB/c background) TPA. A slightly different dosage between the background strains was chosen due to the well-known difference in susceptibility to chemical skin carcinogenesis across strains21. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms12080 For isolation of epidermal single cell suspensions, the skin was ﬂoated dermal side-down in ﬁlter-sterilized, TrypLE Express (Life) solution for 2 h at 37 C, 5% v/v CO2. Following digestion, epidermis was separated from the dermis and further digested in TrypLE Express solution supplemented with 200 mg ml 1 DNAse I (Roche) and 1 DNAse buffer (1.21 g l 1 Tris Base, 0.5 g l 1 MgCl2 and 0.073 g l 1 CaCl2) on a rotator for 30 min at 37 C. Cell suspensions were ﬁltered and washed in PBS before ﬂow cytometry staining. Flow cytometry staining. Cell suspensions were stained with a ﬁxable, live/dead discrimination dye (Life) and subsequently blocked for non-speciﬁc binding using antibody against FcgR (2.4G2) and 2% normal rat serum (Sigma). For staining of cell surface markers, cell suspensions were stained with ﬂuorochrome-conjugated antibodies and appropriate isotype controls for 40 min and subsequently washed. For intracellular cytokine staining cells were incubated for 4 h with brefeldin A at 10 mg ml 1, with or without PMA and ionomycin (6.25 ng ml 1 and 3.5 mg ml 1, respectively). Following stimulation, cells were washed in PBS, resuspended and ﬁxed/permed with Fixation and Permeabilization Buffer Set (Affymetrix, CA, USA) before antibody labelling. For intranuclear gH2AX staining, cells were ﬁxed/ permed in ice-cold 70% ethanol at 20 C for 2 h, then blocked with 2% normal mouse serum, Fc-block and 2% fetal calf serum for 15 min, followed by 45 min staining for gH2AX at room temperature. Stained cells were analysed immediately using a BD FACSVerse (BD Biosciences, NJ, USA) machine. Data analysis was performed using FlowJo 10 for Mac (TreeStar, OR, USA). Antibodies were sourced from eBioscience unless otherwise stated. The following antibodies were used: CD45 (30-F11), Cytokeratin 5 (D516B4; Millipore), IFN-g (XMG1.2), IL-13 (eBio13A), IL-13 Ra1 / CD213a1 (13MOKA), TCR Vg5 (536; BioLegend), gH2AX (JBW301; Millipore), CD64 (X54-5/7.1; BioLegend), CD11c (HL3), CD11b (M1/70), CD207 (4C7; BioLegend), CD103 (2E7) and XCR1 (ZET; BioLegend). Primary neonatal KC cultures. Body wall skin from neonatal mice was incubated overnight at 4 C in 5 U ml 1 Dispase (BD) solution supplemented with 1 antibiotic and antimycotic solution (Sigma). The epidermis was isolated and further digested in TrypLE Express solution supplemented with 200 mg ml 1 DNAse I and DNAse buffer. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms12080 Despite this, the FVB background remained far more susceptible and developed a higher tumour load. All experiments on mutant mice were controlled with the appropriate background strain. Hair regrowth during experiments was gently removed by clipping with trimmers. Mice were monitored daily and cutaneous tumours were counted and measured with a caliper once weekly. Back skin and tumours were evaluated by visual inspection by an observer blinded to the experimental groups. Immunohistochemistry. A 1 1-cm skin cut from the centre of the ears were ﬁxed in formalin, embedded in parafﬁn and 5-mm sections cut. Slides were de- waxed, antigen retrieval was performed with pH 6.0 sodium citrate treatment at 95 C for 15 min, endogenous tissue peroxidase activity was blocked with H2O2 treatment, and non-speciﬁc binding was blocked with normal serum and ﬁsh skin gelatin (Sigma). Non-speciﬁc avidin and biotin-binding sites were blocked with an Avidin/Biotin blocking kit (VECTASTAIN). For BrdU detection, slides were stained with anti-BrdU antibody (clone BU1/75; Abcam) in conjunction with a three-layer immunohistochemistry kit (VECTASTAIN Elite ABC kit; Vectastain) as per the manufacturer’s instructions. Staining was visualised with DAB substrate and brown chromogen precipitation. Slides were counter stained in Harris’ haematoxylin acid solution, dehydrated and mounted with Pertex mouting. Visual inspection of tissue and counting of BrdU þ cells were performed while blinded to the experimental groups. qRT–PCR and primer sequences. RNA was extracted from ammonium-ﬁxed epidermis with an RNEasy Mini kit (Qiagen). RNA was dissolved in nuclease-free water, and yield and purity were determined. Complementary DNA (cDNA) was synthesised from RNA with a iScript cDNA synthesis kit (Bio-Rad) as per the manufacturer’s instructions. cDNA was diluted in nuclease-free double-deionized water for qRT–PCR. All primers were single-stranded DNA oligonucleotides (Sigma) that were intron-spanning as veriﬁed by NCBI Primer-Blast tool. Real-time PCR product was detected with SYBR Green (Life) measured continuously with a ViiA 7 Real-Time PCR system (Applied Biosystems, CA, USA). Ct values for genes of interest were normalised against Ct values of the housekeeping gene Cyclophilin (Cyc) using the 2 DCt method. The following primers were used. F denotes forward primer (50–30) and R denotes reverse primer (30–50). Il13 (F: 50-GCTTATTGAGGAGCTGAGCAAC A-30, R: 50-GCCAGGTCCACACTCCATA-30); Il4ra1 (F: 50-CCAATCAGACAG ATACCAGATG-30, R: 50-CCAGGTCAGCAGCCATTC-30); Il13ra1 (F: 50-AGAG GTTGAAGAGGACAAATGCC-30, R: 50-GCGACAAAGACTGGAATGGTGA G-30); Il13ra2 (F: 50-CCGAAATGTTGATAGCGACAGC-30, R: 50-CCAAGCCCT qRT–PCR and primer sequences. RNA was extracted from ammonium-ﬁxed epidermis with an RNEasy Mini kit (Qiagen). NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms12080 NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms12080 CATACCAGAAAAAC-30); Tslp (F: 50-TCGAGGACTGTGAGAGCAAG-30, R: 50-TGTTTTGTCGGGGAGTGAA-30); Il1a (F: 50-TTGGTTAAATGACCTGCAA CA-30, R: 50-GAGCGCTCACGAACAGTTG-30); Tnfa (F: 50-ACTGGAGTTGTA CGGCAGTG-30, R: 50-GGCTGATCCCGTTGATTTCC-30); Casp3 (F: 50-GAGCT TGGAACGGTACGCTA-30, R: 50-50-GCGAGATGACATTCCAGTGC-30); Il33 (F: 50-CACATTGAGCATCCAAGGAA-30, R: 50-AACAGATTGGTCATTGTA TGTACTCAG-30); Rae1 (F: 50-TGGACACTCACAAGACCAATG-30, R: 50-CCC AGGTGGCACTAGGAGT-30); Claudin1 (F: 50-50-GCCATCTACGAGGGA CTGTG-30, R: 50-50-CACTAATGTCGCCAGACCTGAA-30); Occludin (F: 50- TTGAACTGTGGATTGGCAGC-30, R: 50-CAAGATAAGCGAACCTTGGCG-30); Tgm1 (F: 50-CCTTGAGCTCCTCATTGGAA-30, R: 50-CCCTTACCCACTGGGA TGAT-30); Cdsn (F: 50-AATGTCCAGCCCGGCATAAA-30, R: 50-CAAGATTCC TGGCAGAATAAGACC-30); K5 (F: 50-CATGTCTCGCCAGTCCAGTG-30, R: 50-GGAACCGCACCTTGTCGATG-30); K15 (F: 50-50-GGAAGAGATCCGGG ACAAA-30, R: 50-TGTCAATCTCCAGGACAACG-30); and Cyc (F: 50-CAAAT GCTGGACCAAACACAA-30, R: 50-CCATCCAGCCATTCAGTCTTG-30). DMBA-induced transversion mutation in hras (codon 61, CAA-CTA) was quantiﬁed with a forward primer, mutant-speciﬁc reverse primer and a custom generated TaqMan 60-FAM-conjugated probe for visualizing product: hras C61CAA-CTA (F: 50-CTAAGCCTGTTGTTTTGCAGGAC-30, R: 50-CATGGCA CTATACTCTTCTA-30; Probe: 50-6FAM-CGGAAACAGGTGGTCAT-MGB-30). examined by ﬂow cytometry (Supplementary Fig. 2). Chimeric mice were used for experiments at Z8 weeks after BM reconstitution. Tissue processing. Ears were collected and split into dorsal and ventral sides. For isolation of intact epidermal sheets, the skin was ﬂoated dermal side-down in 0.5 M NH4SCN for 40 min, 37 C, 5% v/v CO2. Epidermal sheets were gently lifted away from the dermis and washed in PBS. The ﬁxed epidermal sheets were then processed either for RNA extraction or for microscopy. Reagents and staining method for epidermal sheets can be found in the Supplementary Information. For isolation of epidermal single cell suspensions, the skin was ﬂoated dermal side-down in ﬁlter-sterilized, TrypLE Express (Life) solution for 2 h at 37 C, 5% v/v CO2. Following digestion, epidermis was separated from the dermis and further digested in TrypLE Express solution supplemented with 200 mg ml 1 DNAse I (Roche) and 1 DNAse buffer (1.21 g l 1 Tris Base, 0.5 g l 1 MgCl2 and 0.073 g l 1 CaCl2) on a rotator for 30 min at 37 C. Cell suspensions were ﬁltered and washed in PBS before ﬂow cytometry staining. Tissue processing. Ears were collected and split into dorsal and ventral sides. For isolation of intact epidermal sheets, the skin was ﬂoated dermal side-down in 0.5 M NH4SCN for 40 min, 37 C, 5% v/v CO2. Epidermal sheets were gently lifted away from the dermis and washed in PBS. The ﬁxed epidermal sheets were then processed either for RNA extraction or for microscopy. Reagents and staining method for epidermal sheets can be found in the Supplementary Information. Methods Mi IL 13 After 21 h, they were injected with 200 ml BrdU solution and 3 h later ear skin was ﬁxed in formalin, embedded in parafﬁn and BrdU staining performed on 5 mm sections. BrdUþ events were counted along the entire length of untreated and treated epidermis. Epidermal BrdU þ cells on the basal membrane were deﬁned as ‘basal’; BrdU þ epidermal cells not on the basal membrane were deﬁned as ‘granular’. Three 1-cm sections were counted per mouse. (h) Number of BrdUþ cells in the basal and granular epidermis at steady state without treatment and (I) 24 h after topical exposure to TPA. (j) The ratio of BrdU þ epidermal cells in the granular layer compared with the basal layer in BALB/c WTand IL-13 / mice. (k) Representative images of ear skin 24 h after exposure to TPA. Scale bar, 50 mm (l) Ear thickness was measured with calipers at 24 h and 72 h after a single skin exposure to 10 nmol TPA on the ear (n ¼ 6 per group). Data are represented as mean±1 s.e.m. 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Real-time PCR product was detected with SYBR Green (Life) measured continuously with a ViiA 7 Real-Time PCR system (Applied Biosystems, CA, USA). Ct values for genes of interest were normalised against Ct values of the housekeeping gene Cyclophilin (Cyc) using the 2 DCt method. Full skin thickness wounding. Mice were anaesthetized with inhalation isoﬂurane and transferred onto a heat block with an anaesthetic nose-cone to maintain anaesthesia throughout the procedure. Buprenorphine local analgesic (Vetergesic) was administered subcutaneously around the area to be wounded. A single, full-thickness 5-mm wound was made at the midline of the lower back using a disposable sterile punch biopsy tool. Sterile paper bedding and wet food were provided for 7 days following wounding. Wound size was measured daily with calipers for the ﬁrst 2 days, and then once every 2 days until complete wound closure. The wound sagittal (x) and transverse (y) plane were measured and these were applied to the ellipse area formula to calculate wound area: (area ¼ p (radius x)(radius y)). The following primers were used. F denotes forward primer (50–30) and R denotes reverse primer (30–50). Il13 (F: 50-GCTTATTGAGGAGCTGAGCAAC A-30, R: 50-GCCAGGTCCACACTCCATA-30); Il4ra1 (F: 50-CCAATCAGACAG ATACCAGATG-30, R: 50-CCAGGTCAGCAGCCATTC-30); Il13ra1 (F: 50-AGAG GTTGAAGAGGACAAATGCC-30, R: 50-GCGACAAAGACTGGAATGGTGA G-30); Il13ra2 (F: 50-CCGAAATGTTGATAGCGACAGC-30, R: 50-CCAAGCCCT Subcutaneous tumour inoculation. The right ﬂank of mice was shaved and 1 104 CT26 cells injected subcutaneously into the ﬂank in 100 ml sterile PBS. The 10 NATURE COMMUNICATIONS \| 7:12080 \| DOI: 10.1038/ncomms12080 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| 7:12080 \| DOI: 10.1038/ncomms12080 \| www.nature.com/naturecommunications Competing ﬁnancial interests: The authors declare no competing ﬁnancial interests. 62. 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USA 104, 6770–6775 (2007). This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ Author contributions 58. Yuspa, S. H. & Morgan, D. L. Mouse skin cells resistant to terminal differentiation associated with initiation of carcinogenesis. Nature 293, 72–74 (1981). T.D. performed and analysed most of the experiments. G.C., M.H. and R.C.R. performed and analysed some experiments. J.S. performed and analysed some experiments, directed the study and wrote the manuscript. 59. Allahverdian, S., Harada, N., Singhera, G. K., Knight, D. A. & Dorscheid, D. R. 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McKenzie for providing IL-13egfp mice, A. Mowat and M. Botto for critical review of the manuscript, O. Sobolev and A. Hayday for insightful discussion and the informed advice of many close colleagues. We gratefully acknowledge the Wellcome Trust for support (100999/Z/13/Z). r The Author(s) 2016 12 NATURE COMMUNICATIONS \| 7:12080 \| DOI: 10.1038/ncomms12080 \| www.nature.com/naturecommunications
https://openalex.org/W2121744252	https://pure.uvt.nl/ws/files/8933671/MCP_Vissers_impact_cancer_diabetes_JoCS_open_access_2015.pdf	English	null	The impact of having both cancer and diabetes on patient-reported outcomes: a systematic review and directions for future research	Journal of cancer survivorship	2,015	cc-by	7,563	The impact of having both cancer and diabetes on patient-reported outcomes Vissers, P.A.J.; Falzon, L.; van de Poll-Franse, L.V.; Pouwer, F.; Thong, M.S.Y. Published in: Journal of Cancer Survivorship DOI: 10.1007/s11764-015-0486-3 Publication date: 2016 Document Version Publisher's PDF, also known as Version of record Link to publication in Tilburg University Research Portal Citation for published version (APA): Vissers, P. A. J., Falzon, L., van de Poll-Franse, L. V., Pouwer, F., & Thong, M. S. Y. (2016). The impact of having both cancer and diabetes on patient-reported outcomes: A systematic review and directions for future research. Journal of Cancer Survivorship, 10(2), 406-415. https://doi.org/10.1007/s11764-015-0486-3 The impact of having both cancer and diabetes on patient-reported outcomes Vissers, P.A.J.; Falzon, L.; van de Poll-Franse, L.V.; Pouwer, F.; Thong, M.S.Y. The impact of having both cancer and diabetes on patient-reported outcomes Vissers, P.A.J.; Falzon, L.; van de Poll-Franse, L.V.; Pouwer, F.; Thong, M.S.Y. impact of having both cancer and diabetes on patient-reported outcomes Citation for published version (APA): Vissers, P. A. J., Falzon, L., van de Poll-Franse, L. V., Pouwer, F., & Thong, M. S. Y. (2016). The impact of having both cancer and diabetes on patient-reported outcomes: A systematic review and directions for future research. Journal of Cancer Survivorship, 10(2), 406-415. https://doi.org/10.1007/s11764-015-0486-3 Citation for published version (APA): Vissers, P. A. J., Falzon, L., van de Poll-Franse, L. V., Pouwer, F., & Thong, M. S. Y. (2016). The impact of having both cancer and diabetes on patient-reported outcomes: A systematic review and directions for future research. Journal of Cancer Survivorship, 10(2), 406-415. https://doi.org/10.1007/s11764-015-0486-3 Citation for published version (APA): Vissers, P. A. J., Falzon, L., van de Poll-Franse, L. V., Pouwer, F., & Thong, M. S. Y. (2016). The impact of having both cancer and diabetes on patient-reported outcomes: A systematic review and directions for future research. Journal of Cancer Survivorship, 10(2), 406-415. https://doi.org/10.1007/s11764-015-0486-3 Tilburg University * Pauline A. J. Vissers p.a.j.vissers@tilburguniversity.edu Abstract Purpose This systematic review aims to summarize the cur- rent literature regarding potential effects of having both cancer and diabetes on patient-reported outcomes (PROs) and to pro- vide directions for future research. Conclusions Having both cancer and diabetes resulted in worse PROs compared to having either one of the diseases, however, the considerable heterogeneity of the included stud- ies hampered strong conclusions. Future studies are needed as this research area is largely neglected. As the majority of the included studies focused on HRQoL, future research should address the impact of both diseases on other PROs such as depression, patient empowerment and self-management. Implications for Cancer Survivor Having both cancer and diabetes might result in worse PROs, however, more research is needed as current evidence is scarce. Methods MEDLINE, The Cochrane Library, CINAHL, and PsycINFO were searched from inception to January 2015. All English peer-reviewed studies that included patients with both cancer and diabetes and assessed PROs were included. All included studies were independently assessed on methodolog- ical quality by two investigators. Results Of the 3553 identified studies, 10 studies were includ- ed and all were considered of high (40 %) or adequate (60 %) methodological quality. Eight of the 10 studies focused on health-related quality of life (HRQoL), functioning, or symp- toms and 2 studies assessed diabetes self-management. Over- all, HRQoL and functioning was lower, and symptoms were higher among patients with both cancer and diabetes as com- pared to having cancer or diabetes alone. Furthermore, one Keywords Cancer . Diabetes . Patient-reported outcomes . Systematic review . 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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 Download date: 24. Oct. 2024 J Cancer Surviv DOI 10.1007/s11764-015-0486-3 REVIEW REVIEW 1 CoRPS—Center of Research on Psychology in Somatic diseases, Department of Medical and Clinical Psychology, Tilburg University, P.O. Box 90153, 5000 LE Tilburg, The Netherlands The impact of having both cancer and diabetes on patient-reported outcomes: a systematic review and directions for future research Pauline A. J. Vissers1,2 & Louise Falzon3 & Lonneke V. van de Poll-Franse1,2 & Frans Pouwer1 & Melissa S. Y. Thong1,2 Received: 16 June 2015 /Accepted: 6 September 2015 # Th A h ( ) 2015 Thi i l i bli h d i h Received: 16 June 2015 /Accepted: 6 September 2015 # The Author(s) 2015. This article is published with open access at Springerlink.co Received: 16 June 2015 /Accepted: 6 September 2015 # The Author(s) 2015. This article is published with ope study reported that diabetes self-management was impaired after chemotherapy. 3 Center for Behavioral Cardiovascular Health, Columbia University Medical Center, New York, NY, USA 2 Department of Research, Netherlands Comprehensive Cancer Organisation, Eindhoven, The Netherlands Introduction Due to the increased aging of the population, early detection, and better treatment of diseases, the number of cancer survi- vors is increasing [1]. As a result, more and more cancer survivors live with other chronic diseases of which diabetes is one of the most prevalent [2]. The prevalence of concurrent diabetes among cancer patients depends on cancer type, gen- der, and age at diagnosis and varies from 8 % among prostate cancer patients to approximately 26 % among pancreas cancer patients aged 65 years or older [2]. This high prevalence of diabetes among cancer patients results in worse outcomes and increases the burden on health systems worldwide. Electronic supplementary material The online version of this article (doi:10.1007/s11764-015-0486-3) contains supplementary material, which is available to authorized users. * Pauline A. J. Vissers p.a.j.vissers@tilburguniversity.edu The link between cancer and diabetes is extensively studied in recent literature and is mainly focused on the impact of diabetes on cancer incidence and mortality. Recent meta- analyses show that diabetes is strongly associated with the J Cancer Surviv development of pancreatic (OR=1.82, 95 % CI: 1.66–1.89) [3], liver (OR=2.50, 95 % CI: 1.80–3.50) [4], and endometrial cancer (RR=2.10, 95 % CI: 1.75–2.53) [5]. Moderate, posi- tive associations have been reported for diabetes and breast (RR=1.20, 95 % CI: 1.12–1.28) [6], colorectal (RR=1.26, 95 % CI: 1.05–1.50) [7], and bladder (RR=1.24, 95 % CI: 1.08–1.42) [8] cancer incidence, while diabetes has been as- sociated with a decreased incidence of prostate cancer (RR= 0.84, 95 % CI: 0.76–0.93) [9]. Furthermore, previous research shows that having diabetes is associated with a 30–40 % in- creased mortality risk among cancer patients, which was mainly apparent among breast, endometrial, and colorectal cancer patients [10, 11]. PubMed related articles were used for the two most recent included studies to identify studies that were not found with the initial literature search. Selection criteria All retrieved studies (including abstracts of unpublished stud- ies) were screened and studies that met the following four selection criteria were included: (1) the study is focused on patients with both cancer and diabetes, (2) PRO is primary or secondary outcome measure of the study, (3) is published in a peer-reviewed journal, and (4) is published in English. Studies that assessed the effects of several chronic or comorbid dis- eases, including diabetes, among cancer patients on PROs were not included as the studies should have a primary focus on both cancer and diabetes. Similarly, studies that aimed to address comorbid or chronic diseases, including cancer, among diabetes patients were excluded. As the group of patients with both cancer and diabetes is growing, patients’ experience of living with both diseases is becoming more important. However, this research area is largely neglected. Patient-reported outcome (PRO) assess- ments such as health-related quality of life (HRQoL), func- tioning, and symptoms are needed as it is plausible that pa- tients with multiple chronic diseases experience more prob- lems. This knowledge is essential to improve clinical practice and care for this growing group of patients. Quality assessment Each selected study was independently scored on methodo- logical quality by 2 reviewers (PV and MT) based on a set of 14 quality criteria (Table 1). These quality criteria were based on established criteria lists used in previous studies [17, 18]. Disagreements between the reviewers on the quality criteria were resolved during a consensus meeting. All studies re- ceived 1 point for each of the 14 quality criteria that was met. If a criterion was not met or described insufficiently, 0 point was assigned. Thus, each study can obtain a maximum score of 14 points. Studies that scored 75 % or more of the maximum attainable score (i.e., ≥11 points) were considered as Bhigh quality study,^ studies scoring between 50–75 % (i.e., 7–10 points) were considered of Badequate quality,^ while those scoring <50 % (i.e., ≤6 points) were considered of Blow quality.^ These criteria were arbitrarily chosen and based on previous research [17]. A significant number of cancer survivors consistently re- port lower physical functioning, sexual functioning, and more symptoms of distress and fatigue [12, 13]. Similarly, diabetes patients are more likely to suffer from depression [14], report a lower quality of life [15], and lower sexual functioning [16]. As both cancer and diabetes patients report deteriorated PROs compared to people without the disease, we hypothesize that having both chronic diseases will result in even more deterio- rated PROs. The aim of this systematic literature review is to summarize the current knowledge on the impact of having both cancer and diabetes on PROs. In addition, as we expect that this research area will be largely neglected, we also aim to provide directions for future research. Description of the included studies LF conducted the systematic literature search on August 2013 and updated the search on January 2015. The following data- bases were included: MEDLINE, The Cochrane Library, CINAHL, and PsycINFO. Subject headings and freetext terms for diabetes (i.e., diabet* OR diabetes mellitus) were com- bined with search terms for cancer (i.e., cancer* OR neo- plasm* OR oncolog). As PROs cover a wide range of differ- ent aspects, we did not include any search terms for PROs to avoid missing relevant papers. The full search strategy is shown in Online Resource 1. After the search was conducted, the cited references of the selected studies were searched using Web of Science and their references lists checked; in addition, The initial broad search strategy on cancer and diabetes that did not include a term for BPROs^ yielded 3553 hits, and after the removal of duplicates and the application of selection criteria, a total of 10 studies were included in this study, of which 2 were based on the same data [19, 20] (Fig. 1). Eight of the included studies had a sample size of at least 590 partici- pants, while 2 studies, based on the same data, included 43 patients [19, 20] (Table 2). The number of patients with both cancer and diabetes was rather low; 5 studies included less than 100 patients with both diseases [19–23]. Moreover, only J Cancer Surviv Table 1 List of criteria for assessing the methodological quality of studies on patient-reported outcomes among patients with cancer and diabetes Positive if with respect to Number of studies that scored positive Patient-reported outcomes N (%) 1. Examining PROs was a primary objective of the study 10 (100) 2. A validated questionnaire to measure PROs was used 10 (100) Study population 3. The patient sampling process is described 10 (100) 4. A (healthy) normative sample is included for comparison 3 (30) 5. Patients with both cancer and diabetes are compared to either patients with only cancer or only diabetes on at least two sociodemographic variables 8 (80) 6. A description is included of at least two clinical variables regarding cancer diagnosis (e.g., cancer stage, treatment, time since cancer diagnosis) 8 (80) 7. A description is included of at least two clinical variables regarding diabetes diagnosis or severity (e.g., HbA1c levels, treatment, time since diabetes diagnosis) 3 (30) 8. Inclusion and/or exclusion criteria are described 9 (90) 9. Description of the included studies Five studies included cancer patients with diabetes (CA+DM+) and made a comparison with cancer patients without diabetes (CA+ DM-) [21, 22, 24, 25, 28], one study compared CA+DM+ patients with patients with diabetes only (CA-DM+) [23] and two studies included CA+DM-, CA-DM+ and patients without both diseases (CA-DM-) for comparison [26, 27]. Two studies, based on the same data, only included CA+ DM+ and did not include a comparison group [19, 20]. Of the 10 included studies, 8 focused on HRQoL, self-perceived health status, functioning, or symptoms, while 2 studies assessed the impact of cancer and its treatment on diabetes self-management. Most studies used a validated question- naire. The Short Form (SF)-36 was used most frequently to assess HRQoL or self-perceived health status [21, 22, 25], other studies used the Health Utility Index Mark 3 (HUI3) [26], the EuroQoL Group’s EQ-5D [23], the Audit of Diabetes Dependent Quality of Life (ADDQoL) [23], the European Organization for Research and Treatment of Cancer core Description of the included studies Participation rates for patient groups are described and these are >75 % 4 (40) 10. Information is given regarding differences in demographic and/or clinical characteristics of respondents vs non-respondents 3 (30) Study design 11. The study sample includes at least 75 patients (arbitrarily chosen) 8 (80) 12. The process of data collection is described 8 (80) 13. The difference in the outcome variable between cancer patients with diabetes and patients with only cancer and/or only diabetes is assessed in multivariable models, including at least 2 covariates 8 (80) Results 14. Mean, median, standard deviations, or percentages are reported and compared between cancer patients with diabetes and patients with only cancer and/or only diabetes for the most important outcome measures 8 (80) Patient-reported outcomes 1. Examining PROs was a primary objective of the study 2. A validated questionnaire to measure PROs was used Study population 3. The patient sampling process is described 4. A (healthy) normative sample is included for comparison 9. Participation rates for patient groups are described and these are >75 % 10. Information is given regarding differences in demographic and/or clinical characteristics of respondents vs non-respondents 11. The study sample includes at least 75 patients (arbitrarily chosen) 12. The process of data collection is described 13. The difference in the outcome variable between cancer patients with diabetes and patients with only cancer and/or only diabetes is assessed in multivariable models, including at least 2 covariates 13. The difference in the outcome variable between cancer patients with diabetes and patients with only cancer and/or only diabetes is assessed in multivariable models, including at least 2 covariates 8 (80) Results 14. Mean, median, standard deviations, or percentages are reported and compared between cancer patients with diabetes and patients with only cancer and/or only diabetes for the most important outcome measures 8 (80) 14. Mean, median, standard deviations, or percentages are reported and compared between cancer patients with diabetes and patients with only cancer and/or only diabetes for the most important outcome measures 8 (80) Quality of Life Questionnaire (EORTC QLQ-C30) [27], or the University of California, Los Angeles, Prostate Cancer Index (UCLA-PCI) [22, 24, 25]. The EORTC QLQ- Chemotherapy-Induced Peripheral Neuropathy (CIPN)-20 was used to assess neuropathic symptoms [28]. Description of the included studies Eight out of 10 studies conducted multivariate analyses and mainly adjust- ed for socio-demographic [19, 21, 22, 24–28] and cancer- related covariates [19, 21, 22, 25, 27, 28], while diabetes- related covariates [19] and lifestyle factors [24, 26–28] were less often adjusted for. Quality of Life Questionnaire (EORTC QLQ-C30) [27], or the University of California, Los Angeles, Prostate Cancer Index (UCLA-PCI) [22, 24, 25]. The EORTC QLQ- Chemotherapy-Induced Peripheral Neuropathy (CIPN)-20 was used to assess neuropathic symptoms [28]. Eight out of 10 studies conducted multivariate analyses and mainly adjust- ed for socio-demographic [19, 21, 22, 24–28] and cancer- related covariates [19, 21, 22, 25, 27, 28], while diabetes- related covariates [19] and lifestyle factors [24, 26–28] were less often adjusted for. 4, of which 3 unique, studies had a longitudinal design [19, 20, 24, 25], while the other 6 studies addressed the associa- tions between cancer and diabetes and PROs cross-sectionally [21–23, 26–28]. Most studies focused on patients with specif- ic cancer types including patients with diabetes and prostate [22–25], colorectal [27, 28], or breast cancer [23]. Five studies included cancer patients with diabetes (CA+DM+) and made a comparison with cancer patients without diabetes (CA+ DM-) [21, 22, 24, 25, 28], one study compared CA+DM+ patients with patients with diabetes only (CA-DM+) [23] and two studies included CA+DM-, CA-DM+ and patients without both diseases (CA-DM-) for comparison [26, 27]. Two studies, based on the same data, only included CA+ DM+ and did not include a comparison group [19, 20]. Of the 10 included studies, 8 focused on HRQoL, self-perceived health status, functioning, or symptoms, while 2 studies assessed the impact of cancer and its treatment on diabetes self-management. Most studies used a validated question- naire. The Short Form (SF)-36 was used most frequently to assess HRQoL or self-perceived health status [21, 22, 25], other studies used the Health Utility Index Mark 3 (HUI3) [26], the EuroQoL Group’s EQ-5D [23], the Audit of Diabetes Dependent Quality of Life (ADDQoL) [23], the European Organization for Research and Treatment of Cancer core 4, of which 3 unique, studies had a longitudinal design [19, 20, 24, 25], while the other 6 studies addressed the associa- tions between cancer and diabetes and PROs cross-sectionally [21–23, 26–28]. Most studies focused on patients with specif- ic cancer types including patients with diabetes and prostate [22–25], colorectal [27, 28], or breast cancer [23]. Study quality The 10 included studies scored a mean quality score of 10 out of 14, and scores ranged between 7 and 13. Four studies (40 %) were classified as being of high quality and 6 (60 %) of adequate quality according to our quality criteria. No studies were considered of low quality. The criteria that were least often met are (#4) the inclusion of a (healthy) normative sam- ple for comparison, (#7) a description of at least two clinical variables regarding diabetes diagnosis, and (#10) information is given regarding differences in demographic and/or clinical characteristics of respondents vs non-respondents (all met by 3 studies) (Table 1). J Cancer Surviv Table 2 Overview of the included studies Study Country Design Study sample Instrument Results Quality score Health-related quality of life/self-perceived health/functioning Bowker et al. (2006) Canada Cross-sectional 113,587 patients with or without cancer (any type) CA+DM+: 207 HUI3 HUI3 score: 10 CA+DM-: 1692 CA+DM+ vs CA-DM-: b=−0.10 (95 % CI: −0.13 to −0.09), P value <0.0001 CA-DM+: 4394 CA-DM+ vs CA-DM-: b=−0.04 (95 % CI: −0.05 to −0.04), P value <0.0001 CA-DM-: 107,295 CA+DM- vs CA-DM-: b=−0.04 (95 % CI: −0.05 to −0.03), P value <0.0001 Hershey et al. (2012a) USA Cross-sectional 661 patients with cancer (any type) CA+DM+: 76 SF-36 Physical functioning: 9 CA+DM-: 585 CA+DM- vs CA+DM+: b=12 (95 % CI: 7–18), P value <0.0001 Latini et al. (2006) USA Longitudinal 1248 prostate cancer patients CA+DM+: 117 UCLA-PCI Urinary function at follow-up: 10 CA+DM-: 1131 CA+DM+ vs CA+DM-: 72±24 vs 77±22, P value=0.01 Mols et al. (2008) TheNetherlands Cross-sectional 590 prostate cancer patients CA+DM+: 65 SF-36 General health: 13 CA+DM-: 525 UCLA-EPCI CA+DM+ vs CA+DM-: b=−0.13, P value <0.01 Vitality: CA+DM+ vs CA+DM-: b=−0.12, P value <0.01 Onitilo et al. (2013) Australia Cross-sectional 3466 diabetes patients either with or without a history of breast or prostate cancer Breast cancer: EQ-5D In men with prostate cancer only: 11 CA+DM+: 77 ADDQoL Problems with mobility: CA-DM+: 1470 CA+DM+ vs CA-DM+: 51 % vs 29 %, P value <0.001 Prostate cancer: Problems in usual activities: CA+DM+: 81 CA+DM+ vs CA-DM+: 35 % vs 25 %, P value=0.035 CA-DM+: 1838 Thong et al. (2011) USA Longitudinal 1811 prostate cancer patients CA+ and incident DM: 215 SF-36 At baseline those with prevalent diabetes report significant lower HRQoL, but after adjustments in longitudinal analyses no differences in HRQoL between CA+DM+ and CA+DM- were observed. Study quality 10 CA+ and prevalent DM: 239 Individual items on BF and SF CA+DM-: 1357 Vissers et al. (2014) TheNetherlands Cross-sectional 2761 patients with or without colorectal cancer and/or diabetes CA+DM+: 328 EORTC-QLQ-C30 Physical functioning 12 CA+DM-: 1731 CA+DM+ vs CA+DM-: beta=−3.8, P value <0.01 CA-DM+: 78 Male sexual problems CA-DM-: 624 CA+DM+ vs CA+DM-: beta=9.4, P value <0.01 Vissers et al. (2015) TheNetherlands Cross-sectional 1193 colorectal cancer patients CA+DM+: 218 EORTC-QLQ-CIPN20 Neuropathic symptoms—tingling fingers or hands 11 CA+DM-: 975 CA+DM+ vs CA+DM-: OR=1.40 (95 % CI: 1.00–1.94) Neuropathic symptoms—tingling toes or feet CA+DM+ vs CA+DM-: OR=1.47 (95 % CI: 1.04–2.07) Neuropathic symptoms—numbness in toes or feet CA+DM+ vs CA+DM-: OR=1.83 (95 % CI: 1.28–2.62) Neuropathic symptoms—males; erection problems CA+DM+ vs CA+DM-: OR=1.83 (95 % CI: 1.11–3.03) J Cancer Surviv Table 2 (continued) Study Country Design Study sample Instrument Results Quality score Diabetes self-management Hershey et al. (2012b) USA Longitudinal 43 patients with a solid tumor and type I or II DM SIC, modified intrusiveness of illness inventory, SCI-R Lower diabetes self-management after 8 weeks on chemotherapy as compared to baseline: 45.86±2.65 vs. 50.84±2.47 7 Higher symptom burden after 8 weeks on chemotherapy as compared to baseline: 32.57±4.49 vs. 25.43 ± 3.81 Overall impact on diabetes self-management was moderate (16.47±8.43), highest impact on: exercise (4.35±2.36), blood sugar monitoring (3.73±2.38) and ability to eat and drink (3.56±2.31) Positive correlation between impact of cancer on diabetes self-management and symptom burden at 8 weeks (r=0.46, P=0.004) Hershey et al. (2014) USA Longitudinal 43 patients with a solid tumor and type I or II DM SIC, DCI, CIDS, OE, HADS, SCI-R Living arrangements, years with DM, total number of medications, baseline DM self-management, DM self-efficacy and baseline and 8-week symptom severity were significant predictors of diabetes self-management. Fig. 1 Flow chart of the selection process of the systematic literature search HRQoL, functioning, and symptoms was regarded clinically relevant [26]. Similarly, lower general health was reported in a cross-sectional study among 65 pros- tate cancer patients with vs 525 without diabetes with average SF-36 scores of 51.9 vs 62.5, which remained significant in multivariable analyses (beta=−0.13) [22]. A longitudinal study among prostate cancer patients did observe differences between CA+DM+ and CA+DM- in general health at base- line, but after adjustments for age, marital status, educational level, income, employment status, baseline HRQoL, cancer stage, primary treatment, baseline PSA, and baseline Gleason score, this difference did not remain significant [25]. Other studies did not report a worse general health among those with both cancer and diabetes [23, 27]. All included studies reported worse PROs among CA+DM+ compared to CA+DM-, CA-DM+, or CA-DM- on at least 1 studied item or subscale, except for 1 longitudinal study [25]. Nine out of the 10 included studies assessed more than 1 PRO, while 1 study only included a general measure of HRQoL [26]. General HRQoL A large cross-sectional study conducted in Canada reported lowest average HRQoL scores for CA+DM+ (n=940) followed by CA+DM- (n=1,692), CA-DM+ (n=4,394), and CA-DM- (n=107,295) patients with average HUI3 scores ranging between 0.67 and 0.89 (i.e., where −0.36=worst pos- sible health, 0=death, and 1=perfect health) [26]. The HUI3 indirectly measures HRQoL using 8 attributes (vision, hear- ing, speech, ambulation, dexterity, emotion, cognition, and pain) and a mean difference of 0.03 was considered as clini- cally important. Multivariable regression analyses showed similar results with a lower HRQoL for CA+DM+, CA+ DM-, and CA-DM+ patients as compared to CA-DM- patients with beta’s of −0.10, −0.04, and −0.04, respectively, which Study quality 1 Flow chart of the selection process of the systematic literature search Cochrane database N=154 Book references N=9 Medline N=2,682 N=10 Articles included Selection criteria include: the study (1) includes patients with both cancer and diabetes, (2) PRO is primary or secondary outcome measure of the study, (3) is published in a peer-reviewed journal, and (4) is published in English Article of our own research group was accepted for publication on the 13th of January 2015 and published online on 3 February 2015 ( ) p g Article of our own research group was accepted for publication on the 13th of January 2015 and published online on 3 February 2015 HRQoL, functioning, and symptoms Study quality 7 ADDQoL Audit of Diabetes Dependent Quality of Life, CIDS Confidence In Diabetes Self-care, DCI Diabetes Complication Index, EORTC QLQ-C30 European Organization for Research and Treatment of Cancer core Quality of Life Questionnaire, EPIC Expanded Prostate Cancer Index Composite, EQ-5D EuroQol Group’s EQ-5D, FLIC Functional Living Index Cancer, HADS Hospital Anxiety and Depression Scale, HUI3 Health Utility Index Mark 3, OE outcome expectancies, SCI-R Self-Care Inventory Revised, SIC Symptoms of Illness Checklist, SF-36 Short Form 36, UCLA-PCI University of California Los Angeles Prostate Cancer Index Quality of Life, CIDS Confidence In Diabetes Self-care, DCI Diabetes Complication Index, EORTC QLQ-C30 European Organization for Research and Treatment nnaire, EPIC Expanded Prostate Cancer Index Composite, EQ-5D EuroQol Group’s EQ-5D, FLIC Functional Living Index Cancer, HADS Hospital Anxiety and Index Mark 3, OE outcome expectancies, SCI-R Self-Care Inventory Revised, SIC Symptoms of Illness Checklist, SF-36 Short Form 36, UCLA-PCI University of I d Table 2 (continued) J Cancer Surviv Selection criteria include: the study (1) includes patients with both cancer and diabetes, (2) PRO is primary or secondary outcome measure of the study, (3) is published in a peer-reviewed journal, and (4) is published in English * Article of our own research group was accepted for publication on the 13th of January 2015 and published online on 3 February 2015 Medline N=2,682 Cochrane database N=154 CINAHL N=621 Psycinfo N=87 Book references N=9 N=3,553 Selected articles N=2,778 Articles screened for content N=775 Duplicates removed N=12 Hardcopies were obtained N=2,766 Studies excluded based on selection criteria* N=8 Articles included N=10 Articles included N=1 Article included as a result of cited reference search N=4 Studies excluded based on selection criteria* N=1 Article included by hand** Fig. 1 Flow chart of the selection process of the systematic literature search Fig. Diabetes self-management DM+ as compared to CA-DM+, but this difference was not found among women with breast cancer [23]. Colorectal CA+ DM+ reported a worse physical functioning as compared to CA+DM- (beta=−3.8) [27]. Two studies did not report lower physical functioning among CA+DM+ [22, 25], however, one study did report lower vitality among prostate CA+ DM+ as compared to CA+DM- (beta=−0.12), which was considered a clinically relevant difference [22]. Two studies, using the same longitudinal data, addressed problems with diabetes self-management among 43 patients with a solid tumor and type 1 or 2 diabetes [19, 20]. One study showed that patients reported higher scores on symptom bur- den and lower scores on diabetes self-management after 8 weeks on chemotherapy as compared to baseline (mean 32.57±4.49 vs 25.43±3.81 and 45.86±2.65 vs 50.84±2.47, respectively) [20]. In addition, a moderate impact of cancer on diabetes self-management was observed, which mainly affect- ed the ability to exercise, blood sugar monitoring, and ability to eat and drink. Moreover, in qualitative assessments many individuals indicated that they prioritized cancer care instead of diabetes care [20]. The other study mainly focused on pre- dictors of diabetes self-management [19]. This study showed that living arrangements, years with DM, the total number of medications, baseline DM self-management, DM self-effica- cy, and baseline and 8-week symptom severity were signifi- cant predictors of diabetes self-management, while diabetes complications, cancer type, stage and treatment, outcome ex- pectancies, and anxiety and depression were not [19]. Neuropathic symptoms A cross-sectional study by our research group among 218 colorectal CA+DM+ and an age- and sex-matched sample of 975 CA+DM- patients assessed differences in neuropathic symptoms. CA+DM+ patients reported more neuropathic symptoms regardless of cancer treatment as compared with CA+DM- patients regarding tingling fingers or hands (OR= 1.40; 95 % CI: 1.00–1.94), tingling toes or feet (OR=1.47 95 % CI: 1.04–2.07), numbness in toes or feet (OR=1.83; 95 % CI: 1.28–2.62), and erection problems among men (OR=1.83; 95 % CI: 1.11–3.03) [28]. However, the majority of reported symptoms were of mild severity. Similar to the results found in our systematic review, liter- ature shows that comorbidity has a significant impact on HRQoL. Several other studies that were not included in this review but included diabetes as one of the studied comorbid conditions showed that cancer patients with comorbidity re- ported lower HRQoL or functioning [30–33]. A few of those studies reported the impact of diabetes separately and found a poorer general health [30], lower physical functioning [30, 33], more symptoms of nausea [31], and more erection prob- lems among CA+DM+ men [32]. In line with these results, Urinary and bowel functioning Three studies among prostate cancer CA+DM+ and CA+DM- patients also focused on prostate cancer-specific symptoms, in- cluding urinary functioning and/or bowel functioning [22, 24, 25]. One study reported lower urinary function during follow- up among prostate CA+DM+ as compared to CA+DM- (mean score 72±24 vs 77±22) [24], but the other studies did not report differences in urinary or bowel functioning [22, 25]. Sexual functioning Sexual functioning was assessed in one study among colorec- tal CA+DM+ [27] and in two studies with prostate CA+DM+ [24, 25]. Colorectal CA+DM+ reported more male sexual problems compared to colorectal CA+DM- (beta=9.4) in a cross-sectional study from the Netherlands [27]. Among pros- tate cancer patients, two longitudinal studies did not observe a significant association between comorbid diabetes and sexual functioning [24, 25]. Discussion The majority of the included studies in this systematic review (i.e., 8 out of 10 studies) addressed HRQoL, self-perceived health, functioning or symptoms, and two studies, based on the same data, assessed diabetes self-management. In all in- cluded studies, CA+DM+ patients reported worse outcomes, but in 1 longitudinal study among prostate cancer patients, differences disappeared after adjustments [25]. CA+DM+ pa- tients mainly scored lower on general HRQoL [22, 26], phys- ical functioning [21, 23, 27], and sexual functioning [27]. In addition, prostate CA+DM+ patients reported lower urinary functioning [24] and lower vitality [22], while colorectal CA+ DM+ vs CA+DM- patients reported more neuropathic symp- toms in a cross-sectional study [28]. Finally, among diabetes patients that also had concurrent cancer, symptom severity increased and diabetes self-management, mainly exercise, blood sugar monitoring, and the ability to eat and drink, was impaired after 8 weeks on chemotherapy [20]. Physical functioning or mobility Five studies included a measure of physical functioning or mobility. In a study with 76 CA+DM+ and 585 CA+DM-, CA+DM+ scored on average 12 points lower on the physical functioning subscale of the SF-36 as compared to CA+DM- [21], as this difference was larger than 0.5 times the standard deviation it can be considered to be clinically relevant [29]. Similarly, a cross-sectional study found more problems with mobility and usual activities among men with prostate CA+ J Cancer Surviv Mental Health CA+DM+ patients did not report worse mental health or emo- tional functioning compared to CA+DM- or CA-DM+ in 3 cross-sectional [21, 22, 27] and 1 longitudinal study [25]. One study included a measure of problems with anxiety, but no significant differences were found between prostate or breast CA+DM+ as compared to CA-DM+ patients in unadjusted analyses [23]. J Cancer Surviv Previous studies show that among both cancer and diabetes patients BMI, physical activity, and smoking are significant predictors of HRQoL [39–43]. However, only 4 of the studies included in this review adjusted for lifestyle factors of which 3 only included BMI [24, 27, 28] and 1 study additionally ad- justed for physical activity and smoking [26]. These studies showed that CA+DM+ patients have a higher BMI [24, 26–28] and are less physically active [26] at baseline than those without diabetes. Although, these studies did observe lower HRQoL among CA+DM+ vs CA+DM- patients inde- pendent of the adjustment for lifestyle factors, more research is needed. It is important to assess whether the poorer lifestyle, rather than clinical factors, of CA+DM+ patients is responsi- ble for the lower HRQoL in this group. Moreover, future re- search should focus on the effect of changes in lifestyle factors and their impact on HRQoL; with that knowledge, interven- tions can be developed to improve HRQoL on the long term. the number of comorbidities, including cancer, among pa- tients with diabetes has also been shown to result in poorer HRQoL [34]. These studies were excluded from the present review as CA+DM+ patients were not the main sample, and as a result the number of included patients with both diseases was often low. Although the included studies were of adequate to high quality, they differed substantially in design, population, and methodology. Different instruments were used to measure HRQoL which hampers comparison of the results. Moreover, different cancer types were studied and sample sizes in sub- groups were generally low, particularly for CA+DM+ pa- tients. The majority of studies included CA+DM+ and CA+ DM- patients, although some studies additionally included a normative sample or CA-DM+ patients for comparison. As a result, information regarding diabetes characteristics was scarce with only 3 out of 10 studies including clinical data regarding diabetes. However, it is important to take the dura- tion and severity of diabetes into account as this may influence the outcomes. Directions for future research Although previous studies suggest that having both cancer and diabetes results in worse outcomes, the evidence is scarce and many relevant topics have not been studied yet. This systematic review shows that the majority of studies focused on general HRQoL and physical function, however, only little attention has been paid to mental health. Mental health was assessed in 5 of the 10 included studies but did not appear to be deteriorated in CA+DM+ patients as compared with CA+ DM- and CA-DM+ patients. However, this might be a result of the used instrument, as all studies used a subscale of a HRQoL instrument, which might not be sensitive to more specific symptoms of anxiety or depression. Depression is a common problem in both cancer and diabetes patients. Previ- ous research shows that depression is highly prevalent, in about a third of all cancer as well as diabetes patients and is associated with worse prognostic outcomes [35–38]. There- fore, it is possible that CA+DM+ patients might encounter more mental health issues, which were not picked up in the limited studies in this review. Thus, future studies should fo- cus on mental health issues, including depression among CA+DM+ patients. Mental Health Only 4 prospective studies were included, of which 2 were based on the same data, and these studies were conducted mainly among prostate cancer patients. Elderly often live with several chronic illnesses such as cancer and diabetes, which poses a burden on patients. Due to the improved survival, self-management of these chronic diseases is becoming more important. This review included two studies on diabetes self-management which showed that cancer patients performed fewer diabetes self-management behaviors, such as monitoring of the blood glucose levels and exercising, after 8 weeks on chemotherapy [20]. More- over, qualitative research showed that diabetes patients who develop cancer prioritize their cancer care over their diabetes care [20]. Among diabetes patients, self-management is wide- ly studied and a previous literature review and meta-analysis shows that self-management interventions can improve blood glucose levels, increase knowledge and self-efficacy, and eventually might reduce costs of healthcare utilization [44]. It is important that both patients as well as specialists recog- nize the importance of self-management of multiple chronic illnesses. It is important that patients are able to utilize their resources and feel that they are in control of life and solve problems when necessary. Therefore, we believe that empow- erment of patients and improving self-management behavior are important topics to address in future studies among pa- tients with multiple chronic diseases. Despite the heterogeneity in patient samples and PROs studied, this systematic review also has several strengths. It is the first to summarize the literature on PROs among CA+DM+ patients. In addition, a broad search strategy was used and thereby a complete overview of the previous literature is pre- sented. Finally, the quality of all included studies was assessed by two independent investigators with a 14-item checklist. Conclusion In conclusion, this systematic review indicates that having both cancer and diabetes results in worse PROs. However, a relatively low number of studies were included and no defin- itive conclusions can be drawn because of the heterogeneity of the included studies. The included studies were of reasonable quality but a main issue was that clinical information regard- ing diabetes was missing. More prospective studies with suf- ficient sample sizes are needed to establish these findings. As this research area is largely neglected and the majority of J Cancer Surviv 11. Renehan AG, Yeh HC, Johnson JA, et al. Diabetes and cancer (2): evaluating the impact of diabetes on mortality in patients with can- cer. 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https://openalex.org/W2987672588	https://link.springer.com/content/pdf/10.1007/s12371-019-00407-9.pdf	English	null	Managing and Surveying the Geological Garden at Tata (Northern Transdanubia, Hungary)	Geoheritage	2,019	cc-by	6,676	Abstract The Geological Garden at Tata is an open-air geological museum where a spectacular succession of Tethyan Mesozoic sedi- mentary rocks, characteristic of the Alpine-Carpathian region, is finely exposed in abandoned quarries and cleaned rock surfaces. In addition to geological values, the area houses copper-age chert mines, the only ones accessible to the wider public in Hungary. Due to financial problems, however, maintenance of the site was more or less confined to mowing for more than 20 years. Renewal of the Geological Garden began in 2015 when large-scale cleaning as well as detailed surveying was carried out. Removing the soil and loose rock debris provided opportunity to study the effects of weathering and growth of vegetation. Near 60 m3 of rock debris and soil had been accumulated on approx. 5000 m2 rock surface over the decades of obligate deterioration. Lower Jurassic marl and Middle Jurassic radiolarite chert beds as well as Middle Jurassic limestones proved to be especially deeply weathered. The cleaning action raised the question whether the sub-horizontal rock surfaces can be conserved for a long time in their present state or not. Lessons drawn from the latter can serve as a basis for future maintenance activities as well as for plans aiming at developing geosites. Keywords Open-air geological museum . Geosite maintenance . Surveying . Peri-Tethyan Mesozoic . Prehistoric mining . Hungary https://doi.org/10.1007/s12371-019-00407-9 Geoheritage (2019) 11:1353–1365 https://doi.org/10.1007/s12371-019-00407-9 Geoheritage (2019) 11:1353–1365 ORIGINAL ARTICLE * István Szente szente@ludens.elte.hu Managing and Surveying the Geological Garden at Tata (Northern Transdanubia, Hungary) István Szente1 & Bence Takács2 & Erzsébet Harman-Tóth3 & Tamás G. Weiszburg1 Received: 10 December 2018 /Accepted: 6 September 2019 # The Author(s) 2019 /Published online: 1 2019 4 November 1 ELTE Tata Geological Garden, Pázmány P. s. 1/c, Budapest 1117, Hungary 2 Department of Geodesy and Surveying, Budapest University of Technology and Engineering, Műegyetem rkp. 3, K ép., Budapest 1111, Hungary 3 Eötvös Museum of Natural History, Pázmány P. s. 1/c, Budapest 1117, Hungary Introduction and Historical Background about the state of geoconservation in Hungary, with special reference to the history, legal background and organization of geoheritage protection. The Act on Nature Conservation (53/ 1996), in force since 1996, provides the basis for protection of “geological formations and scientifically important geological exposures as well as of major localities of rare minerals or fossils” (https://net.jogtar.hu/jogszabaly?docid=99600053. TV). Nowadays, the Ministry of Agriculture holds responsibility for nature conservation and maintains a public database of protected geosites, available only in Hungarian. According to it, about three-fourths of the 195 important geo- logical exposures located in areas protected by a specific law, such as national parks, landscape protection areas and nature conservation areas, are Palaeozoic and/or Mesozoic suites (http://www.termeszetvedelem.hu/index.php?pg=menu_ 2237). Another 88 geosites are protected by a specific law as nature monuments, about 28 of which are also outcrops of pre- Neogene rocks (http://www.termeszetvedelem.hu/orszagos- jelentosegu-egyedi-jogszaballyal-vedett-termeszeti- teruletek). The number of Palaeozoic and Mesozoic exposures located in protected natural areas of local interest is around ten. Abandoned quarries receive less attention than The territory of Hungary, although characterized by surface rocks and sediments of Neogene and Quaternary age, is rela- tively rich in scientifically important and/or spectacular geosites representing earlier periods of Earth history, and some of them were given statutory protection long ago. Recently, Horváth and Lóczy (2015) gave a detailed overview This article is part of the Topical Collection on Geoheritage and Conservation: Modern Approaches and Applications Towards the 2030 Agenda, IX ProGEO Symposium, Poland, 25-28th June, 2018 2 Department of Geodesy and Surveying, Budapest University of Technology and Engineering, Műegyetem rkp. 3, K ép., Budapest 1111, Hungary 3 Eötvös Museum of Natural History, Pázmány P. s. 1/c, Budapest 1117, Hungary 3 Eötvös Museum of Natural History, Pázmány P. s. 1/c, Budapest 1117, Hungary 1354 Geoheritage (2019) 11:1353–1365 underground mines if their protection and conservation is considered, as a rule (Storemyr 2006). In Hungary, however, they are well represented among protected geosites. acknowledged nineteenth century Austrian geologists. Franz Ritter von Hauer and Carl Ferdinand Peters recog- nized the presence of Lower Jurassic red ammonite lime- stone and Upper Triassic Dachstein Limestone, respective- ly (von Hauer 1853; Peters 1859). Lower Cretaceous (i.e. Aptian) beds lying unconformably on Upper Jurassic rocks were discovered by the distinguished Hungarian geogra- pher and geologist Lajos Lóczy sen. (Lóczy 1906). The first detailed account on the stratigraphy of the Kálvária Hill was given by Koch (1909). Introduction and Historical Background Observations were made mostly in three quarries, called “Whitestone”, “Redstone” and “Bluestone”, operating those times (Fig. 2). Many of the most important geosites are concentrated in the Transdanubian Range, where Mesozoic and Cenozoic se- quences are not metamorphosed and usually have not suffered considerable post-depositional deformations. Most of the expo- sures are, however, scattered and far from roads and settle- ments. One of the few exceptions to this rule is the town Tata, situated at the foot of the Gerecse Mountains around 70 km to the west of Budapest (Fig. 1). There a finely exposed succes- sion of Mesozoic sedimentary rocks characteristic of the Alpine-Carpathian region can be studied in abandoned quarries and cleaned rock surfaces of the Kálvária Hill (Calvary Hill, if translated), a fault-bounded rocky horst of around 150 m alti- tude above sea level (Fülöp 1976; Haas 2007). The 1950s of the twentieth century saw a renewed interest in the Mesozoic of the Kálvária Hill, resulting in the compre- hensive papers of Fülöp (1954) and Szabó (1961). József Fülöp (1928–1994), an almost plenipotentiary actor in geolo- gy from the early 1950s to the end of 1980s, i.e. the time of changing in society in Hungary, had the opportunity to make large rock surfaces clean in order to study Middle and Upper Jurassic rocks that have never been quarried being unsuitable for building. Detailed geological survey carried out by him has lead to the recognition of scientific and educational im- portance of the Kálvária Hill Mesozoic succession, and a part of the hill was declared to be a nature conservation area in 1958. In the course of geological study, two chert mining pits dug by the Copper Age man in Middle Jurassic radiolarite were discovered in the late 1960s (Fülöp 1973). The pits, now protected by an exhibition building, are the only prehis- toric mining sites accessible to visitors in Hungary. In addition to the geological and archaeological exploration, Fülöp in- tendedly converted the dusty abandoned quarries into a garden Tata and its environs are relatively poor in natural build- ing stones. With the exception of occurrences of Pleistocene freshwater limestone, hard rocks have been found to crop out almost exclusively on the slopes of the Kálvária Hill, located west of the Lake Öreg (Old Lake, if translated). The variegated Triassic, Jurassic and Cretaceous limestones aroused the interest long ago and were extensively quarried for centuries. Introduction and Historical Background The Lower Jurassic red limestone, often called “marble” is far the most conspicuous of them and has been especially widely used. Robert Townson, an English traveller and nature historian who visited Tata (that time called Dotis or Totis) in 1793, characterized it as “a town built upon a rock of variegated red marble” (Townson 1797). Scientific study of the Mesozoic succession began with the pioneering work of Fig. 1 Location of the Tata Geological Garden 1355 Fig. 2 Geological map of the Kálvária Hill and adjacent areas with location of the exposures mentioned in this paper. Cenozoic formations are not shown except Pleistocene freshwater limestone (after Fülöp 1976, modified) Geoheritage (2019) 11:1353–1365 Fig. 2 Geological map of the Kálvária Hill and adjacent areas with location of the exposures mentioned in this paper. Cenozoic formations are not shown except Pleistocene freshwater limestone (after Fülöp 1976, modified) Geoheritage (2019) 11:1353–1365 1355 Fig. 2 Geological map of the Kálvária Hill and adjacent areas with location of the exposures mentioned in this paper. Cenozoic formations are not shown except Pleistocene freshwater limestone (after Fülöp 1976, modified) in every sense. As a result of this effort, the Geological Garden now houses a wealth of botanical values. 2018). Its extent, visibility and accessibility (corresponding to “potential use values” in Beranová et al. 2017) make the Geological Garden one of the most valued Hungarian geosites. Quarrying came to an end completely in the late 1970s. By this time, the extent of the protected area had increased step-by-step to 3.5 ha and since 1976, it acts as an open-air geological museum, founded by the former Hungarian Geological Institute. The management of the site, now called at full-length “ELTE Tata Geological Garden— Nature Conservation Area and Open-Air Geological Museum”, was taken over by the Eötvös University in 1994. Since then, it functions as a place for public outreach, recreation and teaching. In addition to its educational and cultural importance, the Kálvária Hill holds scientific values and is considered as a geosite sensu Brilha (2016, Economic problems related to the democratic transforma- tion in Hungary dramatically influenced the history of the conservation of geosites including that of the Geological Garden. Until 1992, technicians from the Geological Institute cleared away the soil and loose rock pieces as well as plants from the rock surfaces in the summer months. Around 1600 man-hours were needed once in 2 years to keep the area clean. In addition to the termination of this service, budgetcutsresultedinthereductionofthestaffmembersfrom 5 to 2, making maintenance rather difficult. Rocky surfaces Geoheritage (2019) 11:1353–1365 1356 becamemoreandmorevegetatedandcoveredwithlooserock pieces and soil. Reconstruction of the Geological Garden be- gan in 2015 in form of a large-scale cleaning and surveying action. Lessons drawn from the latter, reviewed below, can serve as a basis for future maintenance activities as well as for plans aiming at developing geosites. formations were studied in detail for the first time on the Kálvária Hill by Fülöp (1976), and type localities of three of them have been designated there. At the Kálvária Hill, an approx. Geoheritage (2019) 11:1353–1365 50-m-thick Upper Triassic to Lower Cretaceous (Aptian) succession is exposed (Fig. 3). The oldest rock exposed is Dachstein Limestone once exploited extensively in the “Whitestone Quarry” located outside the Geological Garden (Fig. 4). Fig. 3 Stratigraphic column of the Mesozoic cropping out at the Kálvária Hill of Tata. (Oxford.– Kimm. Oxfordian– Kimmeridgian, Se. Series, L. Lower, M. Middle, U. Upper, Sy. Systems, Cret. Cretaceous) (after Haas 2007, modified) Stratigraphy and Significance of the Kálvária Hill Mesozoic Succession Macro- and microfossils indicate a Rhaetian, i.e. Late Triassic age of it. A wide variety of sedimentary envi- ronments ranging from areas exposed sub-aerially at times to lagoons of some metre depth are represented in the succession. Beds deposited in lagoons are the most frequent and contain abundant megalodontid The Mesozoic of Tata is described in the comprehensive and abundantly illustrated monograph by Fülöp (1976). Due to the favourable outcrop conditions several Fig. 3 Stratigraphic column of the Mesozoic cropping out at the Kálvária Hill of Tata. (Oxford.– Kimm. Oxfordian– Kimmeridgian, Se. Series, L. Lower, M. Middle, U. Upper, Sy. Systems, Cret. Cretaceous) (after Haas 2007, modified) 1357 Geoheritage (2019) 11:1353–1365 Fig. 4 The “Whitestone Quarry” exposing Dachstein Limestone overlain by Lower Jurassic limestone (Pisznice Limestone) and Rhaetomegalodon incisus, an age-diagnostic (Rhaetian) bivalve from the former one. The specimen is approx. 8 cm high. The dashed line marks the Triassic–Jurassic boundary bivalves (Fig. 4), studied in detail by Végh-Neubrandt (1982). magnificently exposed in the wide quarry wall, i.e. the former “Redstone Quarry”, of the Geological Garden (Fig. 5). The “red marble” is exploited in the Gerecse Mountains since cen- turies, leaving a plenty of abandoned quarries hidden in the forest. Although named after a quarry situated in the northern Gerecse Mountains, Törökbükk Limestone has been intro- duced by Fülöp (1976) on the basis of the “Redstone Quarry” section. The boundary between the Triassic and Jurassic is a flat erosion surface truncating megalodontid bivalves at some places. Dachstein Limestone is overlain by pink then red fine-grained Jurassic limestone beds assigned to the Pisznice Limestone Formation of Hettangian and Sinemurian age. The Middle Hettangian age of the onset of Jurassic sedimentation was refined recently by Pálfy et al. (2007). The Pliensbachian Stage is represented by an intensively bioturbated encrinite, i.e. a crinoidal limestone known as Törökbükk Limestone Formation. The forementioned formations, usually referred to as “Gerecse red marble” in the older literature, are The “red marble” is more than 30 m thick, while cumula- tive thickness of the younger Jurassic strata does not reach 15 m. The Törökbükk Limestone Formation is followed by a less than 1-m-thick clayey red succession known as Kisgerecse Marl. This latter unit of Toarcian age is rather Fig. 5 The 100 m wide and around 15 m high quarry wall exposing an undisturbed Upper Triassic–Lower Jurassic limestone succession is far the most salient element of the view of the lower yard of the Geological Garden. A segment of the wall appearing as an oblique darker band in the photograph has not been cleaned in order to display the state of the wall before the cleaning action of 2015 as well as to study the effects of weathering and the growth of vegetation 1358 Geoheritage (2019) 11:1353–1365 poorly exposed on the surface due to its friable nature. Near the eastern end of the ENE–WSW striking large quarry wall, a normal fault running approximately parallel with it dissects the largely undisturbed Upper Triassic–Lower Jurassic (Pliensbachian) succession. Geoheritage (2019) 11:1353–1365 Middle and Upper Jurassic as well as Lower Cretaceous beds can be studied in excellent expo- sures of more than 2500 m2 extent some meters higher, in the upper terrace (Figs. 6 and 7). forming the Lókút Radiolarite Formation. The material of the minute siliceous tests of radiolarians was usually dissolved during diagenesis and has been precipitated as chert layers and nodules later exploited by the Late Neolithic men. The basal member of the Upper Jurassic is a peculiar sed- imentary breccia bed of some tens of centimetres in thickness, forming a conspicuous marker horizon covering the radiolarite much less resistant to surface weathering (Fig. 8). The older part of the Middle Jurassic series, attributed to the Tölgyhát Limestone Formation, is proved to be consider- ably diverse in facies: red, marly limestone rich in Fe–Mn oxide nodules, crinoidal layers as well as beds containing small-sized bivalve shells in rock-forming quantity occur. In the second half of the Middle Jurassic, the nature of sedimen- tation changed fundamentally: accumulation of calcium car- bonate was largely replaced by silica-rich deposits now The origin of the enigmatic “Oxfordian Breccia” (also known as “Oxfordian Bed”), a widespread member in the Gerecse Jurassic characterized in detail by Fodor and Főzy (2013), is still to be found. According to Fodor and Főzy (2013), it represents most probably a single depositional event, e.g. gravity-driven re-deposition of lime mud. On the other hand, δ13C values usually show a systematic shift through the bed, indicating deposition over an extended Fig. 6 A stairway formed by and made of Lower Jurassic crinoidal limestone connects the lower and upper yards of the Geolo has been taken from the wall of the neighbouring Jewish Cemetery Fig. 6 A stairway formed by and made of Lower Jurassic crinoidal limestone connects the lower and upper yards of the Geological Garden. The photo has been taken from the wall of the neighbouring Jewish Cemetery Geoheritage (2019) 11:1353–1365 1359 Geoheritage (2019) 11:1353–1365 Fig. 7 The upper cleaned rock surface, exposing an eastward dipping Jurassic (Pliensbachian to Oxfordian) succession, with the exhibition hall built above the prehistoric chert pits in the background. Flags were placed to indicate the faults and other phenomena interesting for the wider public on the public outreach of the closing ceremony of the cleaning project in August of 2015 Tithonian bivalves were described by Szente (2013). Unfortunately, the rich and well-preserved Upper Jurassic am- monite assemblage collected bed-by-bed and studied by Gyula Vigh has remained documented only in form of faunal lists published in Fülöp (1976) until now. period (Price 2013). Higher parts of the Upper Jurassic as well as the lowermost Cretaceous are developed in a thin succes- sion of pelagic limestone. Bedding planes of the condensed Pálihálás Limestone of Kimmeridgian age appear as “ammo- nite pavements” and are highlights of the Geological Garden. The Jurassic/Cretaceous boundary can be drawn within the Calpionellid-rich Szentivánhegy Limestone. The latter repre- sents the Tithonian, Berriasian and partly Valanginian stages and has been named after the medieval settlement (Szentivánhegy) once located on the Kálvária Hill, that time called Szentiván Hill. Jurassic/Cretaceous boundary beds were studied in detail recently by Szinger et al. (2007) and The present-day area of the Transdanubian Range was de- formed during the Berriasian to Barremian Ages of the Early Cretaceous, due to the first Eoalpine compressional phase. As a result, a submarine high located in its central part, including the environs of Tata, was formed (Tari 1994; Budai et al. 2018). There, the more or less continuous marine sedimenta- tion that began in the Olenekian Age of the Early Triassic and Fig. 8 Radiolarite chert layers overlain by the “Oxfordian Breccia” (marked with “OB”), a characteristic member of the Kálvária Hill Jurassic Fig. 8 Radiolarite chert layers overlain by the “Oxfordian Breccia” (marked with “OB”), a characteristic member of the Kálvária Hill Jurassic Geoheritage (2019) 11:1353–1365 1360 Fig. 9 Angular unconformity between Upper Jurassic– lowermost Cretaceous limestone beds and Tata Limestone in the “Bluestone Quarry”. The man and the wheelbarrow are standing on the uneven bedding surface of the Szentivánhegy Limestone. Gently dipping beds of Tata Limestone are exposed behind him. U f i f i i di d lasted for more than 110 million years until the Valanginian Age of the Cretaceous was interrupted. Fig. 9 Angular unconformity between Upper Jurassic– lowermost Cretaceous limestone beds and Tata Limestone in the “Bluestone Quarry”. The man and the wheelbarrow are standing on the uneven bedding surface of the Szentivánhegy Limestone. Gently dipping beds of Tata Limestone are exposed behind him. Unconformity surface is indicated by arrow Geoheritage (2019) 11:1353–1365 Cretaceous rocks younger than Tata Limestone, although encountered in trenches, wells and bore- holes to the north-east and south-east of the Geological Garden, are known only from the sub-surface. Near 60 m3 of rock debris and soil were found to be nec- essarytoremove.Thus,alittlebitmorethan1-cm-thicklayer had to be removed as an average. The actual amount, how- ever, significantly depended on the dip of rocky surface and, principally, on lithology. Toarcian marl and Bathonian– Callovian radiolarite beds as well as Middle Jurassic lime- stones were found especially deeply weathered. The subsidy contract has excluded the opportunity to take over any mate- rial from the area of the Geological Garden. A part of the debris, removed formation-by-formation, has been stored in waggons and is available to visitors as samples of local rocks. Radiolarite chert, i.e. the raw material of prehistoric tools, proved to be a popular souvenir since then, especially among children. Fossils gathered during the cleaning action are housed in the collection of the Geological Garden. The remaining bulk of the material has been used to construct a new ramp making accessible a spectacular exposure of Lower Jurassic crinoidal limestone (Fig. 12). Geoheritage (2019) 11:1353–1365 Structural and sedi- mentary evolutions of the Gerecse Mountains and its environs including Tata were studied in detail recently by Fodor (2013). On the Kálvária Hill, approximately 20 million years, representing the Late Valanginian, Hauterivian, Barremian and Early Aptian Ages, is not recorded in rocks. The sedimen- tation, renewed around 115 million years ago in the Clansayesian Subage of the Late Aptian, led to the deposition of the Tata Limestone, a formation widely distributed in the Transdanubian Range between Tata and the town of Sümeg in the west. It overlies the eroded surface of tilted Upper Jurassic limestone beds (Fig. 9). The surface is encrusted by a spectac- ular, 1–2-cm-thick brownish-yellow layer of presumably mi- crobial origin. Fig. 10 Aptian (Lower Cretaceous) ammonites from the basal layers of the Tata Limestone. (a) Parahoplites melchioris. (b) Valdedorsella getulina. (c) Cheloniceras cornuelianum. (d) Holcophylloceras guettardi. (e) Tetragonites duvalianus Tata Limestone, once exploited in the “Bluestone Quarry” at the Kálvária Hill, is the youngest known example of the vanished lithofacies “regional encrinite” sensu Ausich (1997), i.e. a crinoidal formation of several tens of meters thickness and several tens of kilometres areal extent, which has no coun- terpart in modern seas. Type sections of the Szentivánhegy and Tata Limestones have been designated in the “Bluestone Quarry” by Fülöp (1976). Tata Limestone is an appropriate building stone but ex- tremely poor in identifiable fossils. In small depressions of the uneven surface of the Upper Jurassic limestone, however, a large amount of fossils, especially ammonites, brachiopods and gastropods, have been found (Fig. 10). The ammonite assemblage was published by Szives (2007). Several new am- monite species, e.g. Constrictoceras foezyi—named after the acknowledged palaeontologist István Főzy—and C. steineri—named after Tibor Steiner, the leading collector Fig. 10 Aptian (Lower Cretaceous) ammonites from the basal layers of the Tata Limestone. (a) Parahoplites melchioris. (b) Valdedorsella getulina. (c) Cheloniceras cornuelianum. (d) Holcophylloceras guettardi. (e) Tetragonites duvalianus Geoheritage (2019) 11:1353–1365 1361 equipments. The final cleaning of the rock surfaces was car- ried out by water-blasting (Fig. 11). of the “Fülöp Aptian Collection”, now retired keeper of the Geological Garden—have recently been described by Szives (2008) from the area. Such limestone bodies full of fossils were encountered at the base of the crinoidal limestone suc- cession proper only at three places in the whole Transdanubian Range. Fig. 11 Water-blasting proved to be very effective in cleaning lichen-covered limestone surfaces such as the ammonite-rich Upper Jurassic bedding-plane exposed in the “Bluestone Quarry” The Cleaning Action of 2015: Observations and Results in Geosite Conservation The right one has been presented by the Eötvös University in 2008 on the occasion of the 50th jubilee of the Geological Garden as a nature conservation area Limestone blocks exploited here were used as plinth of the Joseph Stalin monument, completed in Budapest in 1951 and torn down in 1956 one has been presented by the Eötvös University in 2008 on the occasion of the 50th jubilee of the Geological Garden as a nature conservation area. Limestone blocks exploited here were used as plinth of the Joseph Stalin monument, completed in Budapest in 1951 and torn down in 1956 Fig. 12 The ramp constructed of rock debris and soil removed during the cleaning action made this small quarry opened in Lower Jurassic crinoidal limestone easily accessible. The left memorial plaque was unveiled in 1969 on the centenary of the Hungarian Geological Institute. The right one has been presented by the Eötvös University in 2008 on the occasion of the 50th jubilee of the Geological Garden as a nature conservation area. Limestone blocks exploited here were used as plinth of the Joseph Stalin monument, completed in Budapest in 1951 and torn down in 1956 established. The network was measured by total station and was adjusted as a free network using GeoEasy soft- ware (Siki 2018). For horizontal and vertical localization, most of the reference points were determined by Real- Time Kinematic Global Navigation Satellite System (RTK GNSS) technique using corrections of the Hungarian GNSS-infrastructure (https://www.gnssnet.hu). As control, independent reference points were also used. Altogether, the network is about centimetre accurate in horizontal and millimetre accurate in vertical sense. At second, the relevant detailed points were measured using traditional tacheometry and total stations. At the end, a digital map with 1:500 scale was drawn in a QGIS project, with layers: nature of the Kisgerecse Marl. In order to prevent the fall of large blocks, two supporting walls have been built (Fig. 13). Another place where building operation, i.e. construction of a supporting pillar proved to be neces- sary, was a part of the wall of the neighbouring Jewish Cemetery built in close proximity to the staircase connecting the lower and upper yards of the Geological Garden. There, a test trenching revealed that the wall has been masoned directly on Upper Miocene sand with no foundation. Both the supporting walls and the pillar have been made of local Lower Jurassic limestone. The Cleaning Action of 2015: Observations and Results in Geosite Conservation In 2014, Eötvös University received a grant of 175,300 € from the European Union in the frame of the programme “Environment and Energy Operational Programme” (Project “Reconstruction of Key Geological Sections at the Tata Nature Conservation Area”). Due to the nature of the admin- istration process related to the project, the work began as late as July of 2015. Thus, largely 2 months was available for the realization of the project instead of the one and a half year originally planned. In addition to cleaning, representative rock surfaces easily accessible to visitors have been polished and infor- mative boards have been placed. Other results of the re- newal include the discovery of a chert mine located out- side of the archaeological exhibition building. The support provided a good opportunity to stop “foresta- tion” and renewal the garden. The work was done by a pro- fessional company, as well as by the staff of the Geological Garden. Plants, soil and loose rock pieces were removed from quarry walls and rock surfaces using hand tools. Flamethrower and environmentally sound defoliant chemicals were also used in the elimination of undesirable vegetation. Steep walls were accessed using ropes and abseiling Constructional operations were found to be necessary in 2015 at two sites. Near its eastern end, the wall of the “Redstone Quarry” dissects a normal fault running large- ly parallel with it. The resulted quarry face exposing stratigraphically younger formations was judged by an expert as unstable and dangerous due to the weakening of rock bodies caused by the fault as well as to the friable Fig. 11 Water-blasting proved to be very effective in cleaning lichen-covered limestone surfaces such as the ammonite-rich Upper Jurassic bedding-plane exposed in the “Bluestone Quarry” 1362 Geoheritage (2019) 11:1353–1365 Fig. 12 The ramp constructed of rock debris and soil removed during the cleaning action made this small quarry opened in Lower Jurassic crinoidal limestone easily accessible. The left memorial plaque was unveiled in 1969 on the centenary of the Hungarian Geological Institute. The right Fig. 12 The ramp constructed of rock debris and soil removed during the cleaning action made this small quarry opened in Lower Jurassic crinoidal limestone easily accessible. The left memorial plaque was unveiled in 1969 on the centenary of the Hungarian Geological Institute. The Cleaning Action of 2015: Observations and Results in Geosite Conservation The project provided opportunity to renew the drainage system of the archaeological exhibition building as well. Previously, the rainwater accumulated on a large part of the upper cleaned surface was thought to be piped through a tube made of asbestos, laid beneath the ground of the exhibition building. In 2015, the old pipe, presumably cracked, was found to be near completely closed up by an 8-m-long tissue of fine roots. The new pipe bypasses the building and cleaning sockets have also been installed. & Land registry maps, as plots, buildings, parcel numbers & Orthophoto made in 2013, available at http://www.fentrol. hu/en/ & Digital terrain model and contour lines & Fences, buildings, inner roads, most important trees & Important geological objects, such as Triassic/Jurassic boundary Results of the Geodetic Survey Surprisingly, the surveying resulted in recognition of consid- erable difference between the extent of the area really occupied by the Geological Garden (3.5 ha) and that indicated by the land registry (2.8 ha). The difference largely lies in the fact that some 0.7 ha, owned by the town, was fenced by the Hungarian Within the 2015 cleaning project, geodetic survey of the whole Geological Garden as well as 3D scan of selected quarry walls and rocky surfaces were carried out. At first, a dense and precise network of reference points was Geoheritage (2019) 11:1353–1365 1363 (1976), indicating that there is no fault between the quarries mentioned. Geological Institute in the 1970s. After recognizing this prob- lem, negotiations were initiated between the stakeholders and since then, the municipality of Tata voted to assign the right of management of the area to Eötvös University. Fig. 13 Due to a normal fault, Lower Jurassic crinoidal limestone beds exposed in the lower half of the wall are in juxtaposition with younger Jurassic formations. The stone masonry partly visible to the right of the wooden scaffolding was built to support the block of thinly bedded Middle Jurassic limestone undercut by the erosion of Toarcian marl (poorly exposed). The former small-shot producing tower, now used as lookout tower, is visible in the background Present-Day Activities and Planning for the Future Results of the geodetic survey and 3D scan are the basis of a digital 3D geological model, whose develop- ment is in progress. Geological results of the surveying include ascertainment of the structure of the rock mass forming the lower yard of the Geological Garden as well. Triassic/Jurassic boundary can be well identified both in the “Whitestone Quarry” and in the quarry situated in the lower yard. The characteristic points of this boundary were measured in three dimensions, and a regression plane was determined on the measured points using GeoEasy software (Siki 2018). The residuals of this re- gression plane are at decimetre level, and the parameters of the regression plane fits the one determined by Fülöp The last 2 years saw a welcome increase in the number of the staff that makes maintenance much more efficient as com- pared to the preceding decades. Since 2016, one of the staff members has been working in the frame of the programme “Workfare work in museums”, coordinated by the Hungarian National Museum, and another one is employed by the local community of Tata. Additional to that, since that year, the botanical values of the Geological Garden have been main- tained by a gardener expert employed by the University. As a consequence of the work carried out by the staff, long 1364 Geoheritage (2019) 11:1353–1365 2002; Stefano and Paolo 2017; Prosser 2019).The Geological Garden well fulfils the criteria of pedagogical geosite and widely used as locale of open-air geological lec- tures. In the last 4 years, altogether more than 1000 students, corresponding to about 15% of the visitors, learned geology over there. unremembered stairways and paths have been re-conquered from the vegetation since 2016. Two more years seem neces- sary, however, to clean up the garden entirely. Recently, how- ever, a new gardening problem has arisen. When forming the garden at the lower yard of the Geological Garden in the early 1980s, different trees have been planted in 1 × 1-m pits sunk into Dachstein Limestone using explosives. Some trees have already outgrown their pits and withered. Finding the fittest species and planting them are tasks for the future. Present-Day Activities and Planning for the Future The Geological Garden of Tata in its renewed form pro- vides visitors a unique opportunity in Hungary to study a spectacular succession of Mesozoic marine sedimentary rocks, as well as prehistoric chert mines, in easily accessible abandoned quarries and other exposures concentrated in a well-groomed garden environment. Keeping rocky surfaces clean, however, would require regular removal of loose rocks, soil and plants. More than 20-year-long pause in this kind of maintenance had resulted in considerable fragmentation of rocky surfaces and lead to the aesthetic deterioration of the Geological Garden. Observations made during the large-scale cleaning action of 2015 clearly indicate that suitably frequent removal of vegetation would be fundamentally important. Obtaining financial assistance to build a visitor centre with a lecture room is another challenging task for the future. The chert pit discovered in 2015 was excavated in 2017 and 2018, in co-operation with the Hungarian National Museum. The majority of the work was done by the enthusi- astic volunteers during the camps organized in the hottest summer weeks by the distinguished archaeologist Katalin Biró-T. The main results of the recent three-year excavation series are three antler tools, reconstructed from chips and de- posited in the Neolithic Collection of the Hungarian National Museum (Bir-T et al. 2018). Although some stratigraphic intervals of the Mesozoic suc- cession of the Kálvária Hill have been studied in very detail in the past, some important questions are still unanswered. The cleaning action served as a good opportunity to collect rock samples from the Middle Jurassic succession whose stratigra- phy is poorly known. Identification of the Tithonian bed from which the calcareous alga Clypeina jurassica was mentioned by Szabó (1961) is also a challenge, this tantalizing record being the first-found evidence of photic zone in the Upper Jurassic of the Transdanubian Range. Acknowledgments Thanks to Zoltán Varga, keeper of the Geological Garden, for his help and hospitality. Thanks to Dóri Farkas, as well, for taking part in the geodetic survey and preparing an excellent diploma thesis. Constructive comments by Ewa Głowniak and Kevin Page editors as well as by three anonymous referees are also gratefully acknowledged herein. Cleaning and surveying work was supported by KEOP-3.1.2/2F/ 09-11 project. Maintenance of the newly excavated chert pit as well as the state of the sub-horizontal cleaned rock surfaces raised the question whether they can be conserved for a long time in their present state or not. Present-Day Activities and Planning for the Future 3D scanning and filling them seem to be an alternative. In that case, visitors could see the rocks lying beneath their feet using VR glasses. Funding Information Open access funding provided by Eötvös Loránd University (ELTE). 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 appro- priate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. In the last years, efforts were made by the management of the Geological Garden to obtain financial assistance for build- ing a visitor centre involving a lecture room necessary for developing new geoeducational programmes. References As in most countries, geology does not appear as an indepen- dent discipline in primary and secondary school curricula in Hungary and is taught in the frame of “Earth and environ- ment”, a subject whose proportion has been reduced in the last decades if the total number of lessons is considered. It is therefore of primary importance to utilize the educational op- portunity provided by the elements of the geological heritage. Geosites, if appropriately selected and maintained, offer espe- cially good opportunities to teach geology to students (Meléndez et al. 2007). Abandoned quarries often serve as valuable resources for education (e.g. Macadam and Shail Ausich WI (1997) Regional encrinites: a vanished lithofacies. In: Brett CE, Baird GC (eds) Paleontological events, stratigraphic, ecological and evolutionary implications. 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https://openalex.org/W4293224283	https://link.springer.com/content/pdf/10.1007/s40544-022-0639-0.pdf	English	null	The mechanisms and applications of friction energy dissipation	Friction	2,022	cc-by	15,821	The mechanisms and applications of friction energy dissipation Huan LIU1, Boming YANG2, Chong WANG1, Yishu HAN1, Dameng LIU1,* 1 State Key Laboratory of Tribology, Tsinghua University, Beijing 100084, China 2 College of Liberal Arts & Sciences, University of Illinois at Urbana-Champaign, Urbana 61801, USA Received: 25 November 2021 / Revised: 06 March 2022 / Accepted: 21 April 2022 © The author(s) 2022. Huan LIU1, Boming YANG2, Chong WANG1, Yishu HAN1, Dameng LIU1,* 1 State Key Laboratory of Tribology, Tsinghua University, Beijing 100084, China 2 College of Liberal Arts & Sciences, University of Illinois at Urbana-Champaign, Urbana 61801, USA Received: 25 November 2021 / Revised: 06 March 2022 / Accepted: 21 April 2022 © The author(s) 2022. Abstract: About 30% of the world’s primary energy consumption is in friction. The economic losses caused by friction energy dissipation and wear account for about 2%–7% of its gross domestic product (GDP) for different countries every year. The key to reducing energy consumption is to control the way of energy dissipation in the friction process. However, due to many various factors affecting friction and the lack of efficient detection methods, the energy dissipation mechanism in friction is still a challenging problem. Here, we firstly introduce the classical microscopic mechanism of friction energy dissipation, including phonon dissipation, electron dissipation, and non-contact friction energy dissipation. Then, we attempt to summarize the ultrafast friction energy dissipation and introduce the high-resolution friction energy dissipation detection system, since the origin of friction energy dissipation is essentially related to the ultrafast dynamics of excited electrons and phonons. Finally, the application of friction energy dissipation in representative high-end equipment is discussed, and the potential economic saving is predicted. Keywords: friction energy dissipation; ultrafast dynamics; origin of friction; superlubricity. Keywords: friction energy dissipation; ultrafast dynamics; origin of friction; superlubricity. Keywords: friction energy dissipation; ultrafast dynamics; origin of friction; superlubricity Keywords: friction energy dissipation; ultrafast dynamics; origin of friction; superlubricity the wear of the flywheel can be reduced, its service quality can be significantly improved. According to statistics, around the world, friction causes ~30% of primary energy waste, and wear causes ~80% of machine parts to fail every year. The average economic loss caused by friction and wear in different countries accounts for about 2%–7% of their GDP [3, 5, 6]. As a major manufacturing country, China accounts for a higher proportion of losses caused by friction and wear. * Corresponding author: Dameng LIU, E-mail: ldm@tsinghua.edu.cn The mechanisms and applications of friction energy dissipation Calculated at only 5% [6], the losses caused by friction and wear may be as high as about 5 trillion RMB in 2020. ISSN 2223-7690 CN 10-1237/TH ISSN 2223-7690 CN 10-1237/TH Friction 11(6): 839–864 (2023) https://doi.org/10.1007/s40544-022-0639-0 https://doi.org/10.1007/s40544-022-0639-0 REVIEW ARTICLE 1 Introduction Tribology is a subject of the science and technology of friction, wear, and lubrication of interacting surfaces in relative motion [1, 2]. It appears in high-end equipment such as microelectromechanical systems, integrated circuits, aerospace, and new energy vehicles. For example, the size of moving parts in the micro-nano devices reaches the order of micro-nano (Fig. 1(a)), and the interface friction there replaces gravity as the dominant force. And the friction and wear interface effect will become the main bottleneck problem, affecting the device’s performance. If car engines worldwide reduce friction by 18% (Fig. 1(b)), 117,000 million in fuel can be saved, and 290 million tons of CO2 emissions can be reduced [3]. The Kepler telescope launched by National Aeronautics and Space Administration (NASA), whose flywheel shaft system failed due to friction (Fig. 1(c)), lost the function of the whole machine after some years of service [4]. If In recent years, the discovery of the superlubricity penomenon has provided a new and vital way to solve the problem of frictional energy consumption [7–17]. In the superlubricity state (Figs. 1(d)–1(f)), the friction coefficient is reduced by orders of magnitude compared with conventional oil lubrication [18, 19]. And the wear rate is extremely low, close to zero. The realization and universal application of the 840 Friction 11(6): 839–864 (2023) Fig. 1 (a) Micro-nano mechanical device, (b) automobile engine, (c) Kepler telescope, (d) schematic diagram of two-dimensional material solid superlubricity, (e) graphene self-recovery phenomenon, and (f) graphene-wrapped tip to achieve superlubricity. Reproduced with permission from Ref. [9] for (a, d), © Springer Nature Limited 2018; Ref. [20] for (e), © American Physical Society 2012; Ref. [7] for (f), © The Author(s) 2017. Fig. 1 (a) Micro-nano mechanical device, (b) automobile engine, (c) Kepler telescope, (d) schematic diagram of two-dimensional material solid superlubricity, (e) graphene self-recovery phenomenon, and (f) graphene-wrapped tip to achieve superlubricity. Reproduced with permission from Ref. [9] for (a, d), © Springer Nature Limited 2018; Ref. [20] for (e), © American Physical Society 2012; Ref. [7] for (f), © The Author(s) 2017. superlubricity will significantly reduce the consumption of energy and resources, and significantly improve the service quality of essential mechanical moving parts. However, the mechanism of superlubricity is still unclear, and existing theories cannot explain some phenomena. The energy dissipation path and mechanism of the friction process are the key to predicting and controlling superlubricity. \| https://mc03.manuscriptcentral.com/friction 2 Classical microscopic mechanism of friction energy dissipation the idea of friction control also stays on controlling the surface roughness and elastoplastic behaviour to influence the friction process. There is still a lack of in-depth research and analysis on the energy dissipation mechanism in the friction process. p p With the development of modern instrument technology and simulation calculation methods, the theoretical system of tribology has gradually entered the micro- and nanoscale. In such scale friction experiments based on the atomic force microscopy (AFM), the researchers found that: (1) Relative sliding occurs between two ideally smooth surfaces, with neither asperities in contact nor sticking, but friction still exists; (2) an atom or atom-level tip is used for a slip experiment on an almost ideal smooth surface. The effect of asperities can be ignored, but there is still stick–slip friction, which consumes energy. At this time, theories such as rough peak contact and plastic deformation in macro-scale friction cannot explain the process of friction energy dissipation at the micro-nano scale, indicating that the mechanism of friction energy dissipation must involve more basic physical processes. Therefore, the origin of friction has once again raised people’s interest. Before suitable detection equipment appears, computer simulation becomes a good research tool. And then, the atomic- level friction model attracts people’s attention, mainly phonon and electron dissipation models for microscopic friction energy dissipation. The PT model regards the interaction on the friction interface as the interplay between the atoms connected by the spring and the periodic potential field. In contrast, it does not consider the interaction between the atoms. In 1938, Frenkel and Kontorova [23] developed a composite vibrator model based on this model. It not only considered the interaction between atoms, but also replaced the periodic potential field with a series of interface atoms connected by a spring network (Fig. 2(b)). This model is also known as the FKT model. Although the composite vibrator model is more complex in form, it does not change the core feature of the transformation of spring deformation energy to vibration energy. In the 1990s, with the rise of molecular dynamics simulation (MDS) technology, researchers can use the above models to analyze the origin of friction at the atomic level [26–30]. Wang et al. [31] and Xu et al. [32] studied the dynamic instability and stick–slip friction behaviour of self-assembled films, graphene, and other systems (Fig. 3(a)). 2 Classical microscopic mechanism of friction energy dissipation The origin of friction has been a long-standing problem. In 1699, Amontons believed that the friction energy loss originated from the contact of the surface asperities when the contact surfaces moved relative to each other. And the elastoplastic behaviours such as meshing and collision between multiple asperities were the cause of friction. In 1785, Coulomb established the theory of unevenness caused by friction and generalized the classical friction law. In 1804, Leslie questioned the theory of friction bumps, thinking that the uphill and downhill would be roughly balanced when two asperities slide through each other [10]. Therefore, for the entire system, there was no change in energy. In the 1950s, Bowden and Tabor [21] proposed the theory of asperity adhesive friction, which assumed that friction originated from the shear and plastic deformation of the adhesive contact point. And they proposed that the friction force is proportional to the actual contact area and the normal load. Although the macro-scale solid friction theory system has become more and more complete, In addition, the friction surfaces in mechanical products not only transfer motion and energy, but also play a decisive role in the efficiency, noise, accuracy, corrosion, reliability, and life of the equipment. New tribological theories and technologies are expected to provide practical solutions to the energy shortage, resource depletion, environmental pollution, and health problems faced by the development of human society. Therefore, we need to reveal the origin of friction and the nature of superlubricity from energy dissipation. Further, exploring the law of frictional energy dissipation is of great significance to high-end equipment manufacturing and the realization of energy conservation and emission reduction. At the same time, it also provides a strong guarantee for achieving the goal of carbon neutrality. Friction 11(6): 839–864 (2023) 841 called the Prandtl–Tomlinson (PT) model [25]. The model states that the frictional upper surface is regarded as non-interacting atoms fixed on rigid support by a spring. The lower surface is regarded as an atomic array with a fixed periodic potential energy field, as shown in Fig. 2(a). The relative sliding of the upper and lower surfaces will trigger the vibration of the atoms on the upper surface. This kind of atomic lattice vibration originating from the friction contact position or spreading to the entire plane is related to the phonon energy dissipation. 2 Classical microscopic mechanism of friction energy dissipation The work showed that the surface structure commensurability and lateral stiffness affected stick–slip friction and energy dissipation. The dissipation form of sliding kinetic energy was mainly caused by the lattice vibration, so the energy is irreversibly dissipated in the form of phonons. Weiss and Elmer [33] studied the dynamic characteristics of 2.1 Phonon dissipation In the research of micro- and nanoscale friction, the first to be developed and perfected is the phonon friction model, including the independent oscillator (IO) model [22] and the Frenkel–Kontorova–Tomlinson (FKT) model [23, 24]. The IO model proposed by Tomlinson in 1929 was the most concerned. Since Prandtl first proposed this model in 1928, it is also Fig. 2 Atomic friction model: (a) PT model, (b) Frenkel–Kontorova model. Fig. 2 Atomic friction model: (a) PT model, (b) Frenkel–Kontorova model. www.Springer.com/journal/40544 \| Friction www.Springer.com/journal/40544 \| Friction 842 Friction 11(6): 839–864 (2023) Fig. 3 (a) Setup of two opposite monolayer films in the MDS. Reproduced with permission from Ref. [31], © Elsevier Ltd 2005. (b) Phonon occupancy number distribution over the frequency range for different temperatures. Reproduced with permission from Ref. [34], © IOP Publishing Ltd. 2009. (c) Friction loop recorded on a boundary region of the sample with adjacent areas of single (1LG) and bilayer (2LG) films. Reproduced with permission from Ref. [42], © The American Physical Society 2009. (d) Friction force as a function of the sliding velocity. Reproduced with permission from Ref. [43], © American Chemical Society 2021. Fig. 3 (a) Setup of two opposite monolayer films in the MDS. Reproduced with permission from Ref. [31], © Elsevier Ltd 2005. (b) Phonon occupancy number distribution over the frequency range for different temperatures. Reproduced with permission from Ref. [34], © IOP Publishing Ltd. 2009. (c) Friction loop recorded on a boundary region of the sample with adjacent areas of single (1LG) and bilayer (2LG) films. Reproduced with permission from Ref. [42], © The American Physical Society 2009. (d) Friction force as a function of the sliding velocity. Reproduced with permission from Ref. [43], © American Chemical Society 2021. found that the friction of monolayer graphene on SiC was larger than that of the bilayer graphene (Fig. 3(c)). They attributed the contrast to the dramatic difference in electron–phonon coupling. Duan et al. [43] found that nonequilibrium phonons could induce resonant vibration of the tip and lead to multiple friction force peaks with the increasing sliding velocity (Fig. 3(d)). the two-atom plane sliding at a constant speed, and pointed out that the friction force would increase under the influence of the coherent motion of phonons. Chen et al. [34] showed the temperature-dependent friction in carbon nanotubes. 2.1 Phonon dissipation They found that phonons would be excited by the mechanical vibration when the temperature reached a critical value, increasing the friction force (Fig. 3(b)). Vink [35] investigated the influence of the phonon back-scattering effect of the substrate on the sliding friction by the MDS. They proved that during sliding friction, the energy of the upper friction pair is mainly transferred to the phonon mode propagating vertically on the substrate. At the same time, they proposed that adjusting the life span of vertically propagating phonons can control friction. \| https://mc03.manuscriptcentral.com/friction 2.2 Electron dissipation [60] found that VO2 exhibited metallic characteristics above a critical temperature, and its internal current and friction increased with the increasing temperature. The increasing current was attributed to the increase in the number of carriers in VO2. However, the increasing friction resulted from the strong Coulomb attractive interaction between the trapped charge and the tip. Park et al. [61, 62] applied different voltages to the pn junction of silicon. They found a strong accumulation of carriers in the p-region with a large ohmic loss when a positive voltage was applied, leading to a significantly higher friction force than that in the n-region (Fig. 5(a)). He et al. [63] found that in-plane carriers could suppress the high-energy dissipation process at the friction interface of MoS2, thereby reducing friction (Fig. 5(b)). Fang et al. [64] used the electron beam injection method to change the carrier concentration in MoS2 for controlling the friction force. In addition, an external conductive layer could modulate the electrical properties of the friction interface [65]. Fig. 4 Diagram of Coulomb drag between two closely-spaced solids. Reproduced with permission from Ref. [50], © The American Physical Society 1998. Fig. 4 Diagram of Coulomb drag between two closely-spaced solids. Reproduced with permission from Ref. [50], © The American Physical Society 1998. In the early years, the research on electronic frictional energy dissipation mainly focused on the changes in macroscopic physical and chemical properties, resulting from the electronic behaviour, such as the increase in interface resistance caused by the film adsorption effect [51–53], the appearance of infrared absorption peaks [54], and the broadening of the vibration peak [55]. Persson et al. [56] and Dou and Subotnik [57] found that the resistivity of the metal film increased when the adsorbent slid on the metal surface. The generated kinetic energy excited the conduction band electrons when the adsorbent moved on the metal surface, resulting in damping at the interface. Witte et al. [58] found a strong interaction between the octane molecules and the clean metal Ru substrate, which excited electron–hole pairs in the Some calculations have also shown that the interfacial electric field can affect the interlayer charge distribution of two-dimensional materials, affecting the interlayer interaction and surface energy barrier and leading to the change of friction behaviour. Wang et al. [66] and Wang et al. [67] studied the influence of electric field on the friction between graphene and MoS2. 2.2 Electron dissipation In addition to phonon excitation, ohmic heat is generated between the sliding surfaces due to the lattice scattering of excited electrons [44, 45]. Friction electron dissipation essentially comes from the frictional Coulomb drag effect between two very close layers. The moving atoms of the top layer can drag the carriers of the bottom layer by Coulomb interaction [46–49]. Therefore, additional friction and ohmic energy loss are generated. Persson and Zhang [50] first used the Coulomb drag model to analyze the In addition, a series of micro- and nanoscale friction experiments have also verified the vibration-based friction phonon dissipation [36–41]. Filleter et al. [42] \| https://mc03.manuscriptcentral.com/friction \| https://mc03.manuscriptcentral.com/friction Friction 11(6): 839–864 (2023) 843 friction force between two atomically smooth metal surfaces separated by a vacuum slab of thickness d (Fig. 4). They found that when the distance is small enough (a few nanometers), even if there is no potential difference between the two surfaces (no electric field at the interface), due to the thermal or quantum fluctuations, an unsteady state of charge distribution will still be generated at certain positions. This unstable charge distribution gives rise to a temporal charge imbalance and a spatial electric field. If the two surfaces slide relative to each other, there is always an energy difference between them. The relatively moving surface will continue to transfer energy to the relatively stationary surface in the form of electron dissipation, triggering currents inside the friction surface or at the contact interface. Finally, the current is dissipated as the ohmic heating under the influence of material resistance. metal Ru substrate when they oscillated into and out of the surface. This implied that the electrons controlled the octane molecule’s vertical vibration and damping motion, leading to the molecular’s vibration peaks broadening. In addition, Highland and Krim [59] compared the friction changes between the lead (Pb) substrate and the nitrogen (N2), helium (He), and water (H2O) molecules before and after the superconductivity, and found that the H2O molecules caused stronger electronic friction dissipation. At present, there is no powerful tool to detect the electron dynamics in the friction process directly. Therefore, researchers apply an external voltage to affect the process of electronic friction dissipation, which can change the concentration and distribution of carriers on the surface of the friction pair. Kim et al. 2.2 Electron dissipation They found that the www.Springer.com/journal/40544 \| Friction 844 Friction 11(6): 839–864 (2023) Fig. 5 (a) AFM measurements on silicon pn junction device. Reproduced with permission from Ref. [62], © The American Association for the Advancement of Science 2006. (b) Atomic-scale friction mappings with and without external electric field. Reproduced with permission from Ref. [63], © WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 2019. (c) Interfacial charge density fluctuation of MoS2/MoS2 heterostructures during sliding. Reproduced with permission from Ref. [68], © The Royal Society of Chemistry 2017. Fig. 5 (a) AFM measurements on silicon pn junction device. Reproduced with permission from Ref. [62], © The American Association for the Advancement of Science 2006. (b) Atomic-scale friction mappings with and without external electric field. Reproduced with permission from Ref. [63], © WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 2019. (c) Interfacial charge density fluctuation of MoS2/MoS2 heterostructures during sliding. Reproduced with permission from Ref. [68], © The Royal Society of Chemistry 2017. simultaneously and couple with each other, which also brings some challenges to the study of electronic friction dissipation. electric field could inject different electron densities, thus controlling the interlayer interaction. Wang et al. [68] showed that the atomic-scale friction was determined by the fluctuation of the sliding-induced interface charge density (Fig. 5(c)). \| https://mc03.manuscriptcentral.com/friction 2.3 Non-contact friction energy dissipation Although the existence of electronic friction dissipation has been fully verified [69–77], its systematic model has not been formed [78–80]. Because the electronic dissipation process is affected by a variety of factors, such as electric energy band structure, carrier distribution state, and charge relaxation time. Moreover, the origin of electron dissipation involves quantum theory, and their related experiments require strict experimental conditions and test accuracy. In addition, in the actual friction dissipation process, the phonon and electron dissipation process often occur The non-contact friction issue has been widely studied because of its simplified model and ultrasensitive force detection. In 1997, Pendry [81] calculated the friction between two infinite, parallel (with very small spacing) and relatively moving with a constant speed. He found that friction still existed even in an environment of absolute zero temperature, resulting from electromagnetic fluctuations in the vacuum. Volokitin and Persson [82] further studied the friction between two metal conductors with tens of nanometers and sliding relatively parallel. Theoretically, when there Friction 11(6): 839–864 (2023) 845 Non-contact friction can be investigated by attaching an extremely sharp tip on an elastic cantilever, which allows to register the strength of the normal and lateral force components between the surface and the tip (Fig. 7(a)). Kisiel et al. [39] studied the dissipation mechanism of phonon and electron in Nb film under non-contact friction conditions. They found that the non-contact friction coefficient Γ, closely related to the probe–sample distance d and the bias voltage Vs, was reduced by a factor of three when the sample entered the superconducting state. Specifically, in the metallic state, Γ was proportional to d−1 and 2 s V ; while in the superconducting state, Γ was proportional to d−4 and 4 s V , as shown in Fig. 7(b). This indicated that electronic friction was the main dissipation channel in the metal state; in the superconducting state, electronic friction was suppressed, and phonon friction became the main dissipation channel. were some adsorbents on the metal surface, they could cause resonance photon tunnelling or low-frequency surface plasma. Thus, the surrounding electromagnetic field had large fluctuations, giving rise to a large increase in the friction between the two metal planes. Although the influencing factors of quantum friction are different, such as distances [83], shapes [84], surface adsorbents [85], and materials [86], the essence is related to the electromagnetic field fluctuation, which in turn changes the friction. 2.3 Non-contact friction energy dissipation There are four microcosmic mechanisms for non-contact friction energy dissipation in Fig. 6 [65, 87–89]. (1) Van der Waals friction: Due to quantum fluctuations with no external field, an electric dipole is induced in the above tip, further inducing a dipole in the bottom surface. When the dipole in the tip emits a photon, its wave is Doppler-shifted in the bottom body, resulting in a different reflection amplitude. The same is true for the second body. The exchange of Doppler-shifted photons is the origin of the van der Waals friction energy dissipation. (2) Electrostatic friction: When an electrical field is applied or an inhomogeneous tip-sample electric field is unavoidable, the induced charges follow the tip motion, leading to Joule energy losses. (3) Phononic friction: The local elastic deformation of the surface resulting from the van der Waals interaction or electrostatic interaction follows the tip motion and induces the energy dissipation in the form of phonons in the substrate. (4) Adsorbate drag friction: When there are adsorbates on the surface, a moving tip will induce a drag force, owing to the van der Waals interaction or electrostatic interaction between the tip and adsorbates. The topological insulator has topologically protected surface electronic states from preventing the back- scattering of electrons, in which Joule dissipation is suppressed. Yildiz et al. [88] combined the scanning tunnelling microscopy (STM) and pendulum atomic force microscopy (pAFM) to perform non-contact friction experiments on the surface of Bi2Te3 in Fig. 7(c). They found that due to the topological protection of the surface state, the relationship between the friction coefficient and the bias deviated from the parabolic shape. And dissipation peaks appeared at some special bias positions, indicating that Joule dissipation was suppressed, as shown in Fig. 7(d). In addition, as the magnetic field intensity increased, which destroyed the topological protection surface state, it is observed Fig. 6 (a) Van der Waals friction, (b) electrostatic friction, (c) phononic friction, and (d) adsorbate drag friction. Reproduced with permission from Ref. [87], © Springer-Verlag Berlin Heidelberg 2017. Fig. 6 (a) Van der Waals friction, (b) electrostatic friction, (c) phononic friction, and (d) adsorbate drag friction. Reproduced with permission from Ref. [87], © Springer-Verlag Berlin Heidelberg 2017. www.Springer.com/journal/40544 \| Friction Friction 11(6): 839–864 (2023) 846 Fig. \| https://mc03.manuscriptcentral.com/friction 2.3 Non-contact friction energy dissipation 7 Non-contact friction experiment with electrons and phonons: (a) pAFM, (b) relationship between non-contact friction coefficient Γ and probe-sample distance d, (c) combined STM–pAFM measurements, and (d) dissipation–bias curve under different applied magnetic fields. Reproduced with permission from Ref. [39] for (a, b), © Macmillan Publishers Limited 2011; Ref. [88] for (c, d), © The Author(s), under exclusive licence to Springer Nature Limited 2019. Fig. 7 Non-contact friction experiment with electrons and phonons: (a) pAFM, (b) relationship between non-contact friction coefficient Γ and probe-sample distance d, (c) combined STM–pAFM measurements, and (d) dissipation–bias curve under different applied magnetic fields. Reproduced with permission from Ref. [39] for (a, b), © Macmillan Publishers Limited 2011; Ref. [88] for (c, d), © The Author(s), under exclusive licence to Springer Nature Limited 2019. gradually realized that the origin of friction is mainly related to the dynamics processes of energy carriers such as phonons, electrons, and photons at the friction interface. These energy dissipation pathways involve the charge transfer, electron–electron interaction, Auger recombination of carriers, lattice vibration, as well as processes such as exciton recombination, diffusion, annihilation, etc. (Fig. 8). These processes usually occur in the femtosecond range to nanosecond [11, 79, 90–94], which is difficult to detect. Thus, to reveal the friction energy dissipation mechanism, it is necessary to systematically study the energy dissipation issue at nanoscale and ultrafast. But the existing instruments can no longer satisfy the requirement. In addition, a real friction interface involves multi- physics coupling such as electricity, heat, force, and light. These complicated conditions have reached the detection limit of existing scientific instruments in the field of tribology and require extremely high space-time challenges for high-sensitivity in-situ detection. that the dissipation peak decreased and Joule dissipation reappeared. This work provides a new idea for regulating non-contact frictional energy dissipation channels. Although the non-contact frictional energy dissipation mechanisms of van der Waals friction, electrostatic friction, phonon friction, and adsorbate drag friction have been confirmed in experiments, their internal mechanisms are still unclear. Taking phonon friction as an example, although we have found that the friction process can excite phonons, how the friction phonons dissipate energy is unknown. This requires further exploration of the types of phonons and their dynamic behaviour, which is the key to clarifying the mechanism of phonons. Therefore, the non-contact frictional energy dissipation mechanism needs to be studied at a more microscopic level. 3.3 Ultrafast energy dissipation detection To detect the energy dissipation of phonons and electrons in the friction process, there are still three major difficulties: (1) Both the phonon and electron dissipation processes occur in an ultrafast time scale (the order of fs–ps). Hence, the experimental detection of phonon and electronic dynamics is a challenge. (2) Solids will not be ideal crystals like the Tomlinson model. The appearance of defects and impurities often accompanies the interface. Especially in the atmospheric environment, covalent bonds between layers and chemical pinning of macromolecule adsorption easily occur in friction interfaces. (3) The actual friction process, in addition to the phonon and electron dissipation, also emits X-rays, visible light, plasma, etc. Moreover, it involves material deformation, molecular orientation and chemical reactions, etc. [117, 118]. The ultrafast spectroscopy is an effective tool for studying the dynamics of carriers or excitons in materials [119–123]. For example, time-resolved fluorescence technology is used to study the radiative dissipation process, and pump-probe technology is used to detect the non-radiative dissipation process. Liu et al. [124] used the fluorescence lifetime imaging system to study the relationship between the exciton radiative recombination lifetime and the layer number in transition-metal dichalcogenides (TMDCs) (Figs. 9(a)–9(c)). Exciton radiative lifetime decreased 3.1 Ultrafast phonon energy dissipation dynamics The friction energy dissipation path is almost all related to the thermal energy carried by various vibrations at the atomic scale [80]. Phonon describes the lattice vibration with different frequencies [43, 95–98]. Then, the phonons dissipate their energy through ultrafast processes such as electron–phonon scattering [99], phonon diphase [100], and exciton–phonon coupling [101], which determine the phonon lifetime. Ding et al. [102] found that the energy accumulated in the adhesion stage and completely dissipated within 100 ps by the excitation of phonons at the Cu interface. Chen’s research group [103] studied the energy dissipation between graphene layers through the MDS. They found that the sliding velocity affected the phonon lifetime, which proved that reducing phonon lifetime or increasing phonon scattering intensity could increase friction. Sakong et al. [104] calculated that the vibrational lifetime of Ge interface under saturated hydrogen was 1.56 ns. But under saturated both hydrogen and deuterium, the tensile vibrational life of Ge–D was six times longer than that of Ge–H. 3 Ultrafast friction energy dissipation With the development of quantum tribology, people 847 Friction 11(6): 839–864 (2023) Fig. 8 Schematic of ultrafast friction energy dissipation process. semiconductor interface materials, the excited electron and hole are bound to form an exciton due to the bandgap and Coulomb interaction. Therefore, the concept of friction electron dissipation process includes carriers and excitons. When the excited state electrons or excitons return to the ground state, energy is released through radiative or non-radiative energy dissipation pathways. These dissipation channels include charge transfer [106], Auger recombination [107], electron–electron interaction [108, 109], and electron–phonon coupling [110] of carriers, as well as processes such as energy transfer [111], radiative recombination [112], exciton–exciton annihilation [113, 114], exciton–phonon interaction, and diffusion [115] of excitons. These processes usually occur in the range of femtosecond to nanosecond [116]. Fig. 8 Schematic of ultrafast friction energy dissipation process. www.Springer.com/journal/40544 \| Friction 3.2 Ultrafast electron energy dissipation dynamics In the friction process, the kinetic energy of the friction pair can excite electrons, holes, and electron–hole pairs (excitons) at the material interface (Fig. 8). In the metal or semiconductor interface, electron dissipation becomes an important form of friction energy dissipation [105]. In metal interface materials, free electrons or holes are called carriers; while in www.Springer.com/journal/40544 \| Friction 848 Friction 11(6): 839–864 (2023) Fig. 9 (a, b, and c) Exciton radiative recombination lifetime of layered MoS2, WS2, and WSe2, respectively. (d) Schematic of the energy transfer process from QDs to MoS2. (e) Time-resolved photoluminescence (PL) decay traces in QDs/h-BN/MoS2 heterostructure with different thicknesses of h-BN spacers. Reproduced with permission from Ref. [124] for (a, b, c), © American Chemical Society 2019; Ref. [125] for (d, e), © WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 2019. Fig. 9 (a, b, and c) Exciton radiative recombination lifetime of layered MoS2, WS2, and WSe2, respectively. (d) Schematic of the energy transfer process from QDs to MoS2. (e) Time-resolved photoluminescence (PL) decay traces in QDs/h-BN/MoS2 heterostructure with different thicknesses of h-BN spacers. Reproduced with permission from Ref. [124] for (a, b, c), © American Chemical Society 2019; Ref. [125] for (d, e), © WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 2019. to nanoseconds. But non-radiative energy dissipation processes often require higher time resolution. For example, the exciton formation and the defect trapping exciton processes are all in the range of 100 femtoseconds to picoseconds, which requires the help of femtosecond transient absorption imaging technology detection that based on pump-probe [126–131], as shown in Fig. 10(a). This technology offers an effective tool to study the ultrafast electron energy dissipation at the nanoscale. Especially in two-dimensional semiconductors, the friction interface is always accompanied by a series of defects [9, 132–137], which often significantly impact excitons’ ultrafast energy dissipation process. Therefore, studying the influence of defects on exciton dynamics provides insight into the friction mechanism [138, 139]. Liu et al. [140, 141] built a femtosecond transient absorption imaging system to study the effect of defects on the process of exciton radiative recombination and annihilation in WS2. The defect captured neutral exciton in 7.75– 17.88 ps to form a long-lived defect-bound exciton (Figs. 10(b)–10(e)). Further, the process of defects hindering exciton diffusion was directly visualized (Fig. 10(f)), which realized the imaging detection of the ultrafast energy dissipation process. It is found that with the TMDCs’ layer number reducing. \| https://mc03.manuscriptcentral.com/friction 3.2 Ultrafast electron energy dissipation dynamics The fastest exciton recombination rate was observed in monolayer TMDCs, attributed to their reduced dielectric screening. Liu et al. [125] studied the charge transfer and energy resonance transfer mechanism in quantum dots/MoS2 heterojunction (Figs. 9(d) and 9(e)), whose structure was similar to the non-contact friction in Fig. 6. When quantum dots (QDs) were directly spun onto the surface of MoS2, the non-radiative energy dissipation pathway in the heterojunction was mainly charge transfer. As the layer number of MoS2 increased, the non-radiative energy dissipation rate decreased. When h-BN was inserted between QDs and MoS2, the non-radiative energy dissipation pathway became the energy resonance transfer. As the thickness of h-BN increased, the non-radiative energy dissipation rate decreased. These results reveal the mechanism of the dielectric property, and layer number and distance on the energy dissipation rate, and realize the controlled energy transfer rate. As mentioned above, the ultrafast electron energy dissipation process can be divided into radiative and non-radiative processes. Time-resolved fluorescence imaging technology is usually used to monitor radiative lifetime in the range of 100 picoseconds 849 Friction 11(6): 839–864 (2023) Fig. 10 (a) Schematic of the femtosecond transient absorption microscopy. (b–e) Defects bind excitons to form defect-induced excitons with a long lifetime. (f) Directly visualization of exciton transport in WS2. Reproduced with permission from Ref. [140] for (a, f), © WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 2019; Reproduced with permission from Ref. [141] for (b–e), © The Royal Society of Chemistry 2019. Fig. 10 (a) Schematic of the femtosecond transient absorption microscopy. (b–e) Defects bind excitons to form defect-induced excitons with a long lifetime. (f) Directly visualization of exciton transport in WS2. Reproduced with permission from Ref. [140] for (a, f), © WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 2019; Reproduced with permission from Ref. [141] for (b–e), © The Royal Society of Chemistry 2019. the defect drastically reduced the exciton diffusion coefficient and the exciton diffusion length. to the contact between the tip and the surface of the sample, the tip interacts with multiple atoms on the surface of the sample, resulting in complex tip–sample interactions. Therefore, it is difficult to achieve accurate analysis. Saitoh et al. [142] and Weymouth et al. [143] used a high-quality factor quartz tuning fork (q-Plus AFM) to detect frictional force and frictional energy dissipation. 3.2 Ultrafast electron energy dissipation dynamics By placing the quartz tuning fork perpendicular to the sample and ensuring that the tip oscillates parallel to the sample, direct detection of frictional force and frictional energy dissipation can be achieved under non-contact conditions. In addition, this device integrates the STM function to spatially achieve atomic resolution. To further improve the resolution of frictional energy dissipation, Kisiel et al. [39] and Langer et al. [144] used a very soft and sensitive cantilever placed perpendicular to the sample surface, so that the 3.4 High-resolution friction energy dissipation detection system Although the above researchers have used the ultrafast spectroscopy to study the energy dissipation process of electrons and phonons at the interface of two-dimensional materials widely used in the field of friction, they have not yet detected ultrafast energy dissipation under friction. At present, some kinds of friction energy dissipation detection instruments play an important role, which includes the AFM, q-Plus AFM, and pAFM. The AFM is widely used in tribology, which detects frictional force and frictional energy dissipation by detecting the torsional deformation of the cantilever caused by the contact sliding of the probe with the surface. Due www.Springer.com/journal/40544 \| Friction Friction 11(6): 839–864 (2023) 850 cantilever oscillated parallel to the surface, which likes a pendulum and is named the pAFM. The pAFM can achieve a friction coefficient resolution of 10−12 kg·s−1 and a frictional energy dissipation resolution of 65 μW. Although these detection instruments have extremely high spatial and energy resolution, they are not time-resolved and cannot effectively detect the ultrafast dynamic process of phonons and electrons in frictional energy dissipation. However, these dynamic processes are the key to a deep comprehension of friction energy dissipation. There is an urgent need for an instrument with high temporal resolution, high spatial resolution, and high frictional energy dissipation resolution at the same time. ultrafast energy dissipation and the measurement of friction energy dissipation in-situ and real-time. The instrument architecture consists of six chambers with an H-shaped layout. In addition, it is composed of vacuum interconnection, multi-level vibration reduction, and a thousand-level clean atmosphere to ensure environmental consistency of scientific experiments under extreme conditions such as ultra-high vacuum and low temperature. The origin of friction energy dissipation and the nature of superlubricity can be explored from the perspectives of the ultra-low friction coefficient (0.0001), the ultrafast dynamics of friction phonon, the detection of the ultra-broad spectrum of tribo-emission in the order of eV, high- speed online measurement of molecular orientation, and structural evolution at friction interface. The successful development of this instrument will provide a powerful test tool for discovering new ways to reduce friction energy After long-term exploration, the research team of Luo [6] has recently developed a friction energy dissipation measurement system (Fig. 11), which has made breakthroughs in basic research such as ultra-high friction coefficient measurement resolution and Fig. 11 (a) Schematic and (b) partial physical mappings of the high-resolution friction energy dissipation detection system. \| https://mc03.manuscriptcentral.com/friction 3.4 High-resolution friction energy dissipation detection system Reproduced with permission from Ref. [6] for (a), © Science China Press 2020. Fig. 11 (a) Schematic and (b) partial physical mappings of the high-resolution friction energy dissipation detection system. Reproduced with permission from Ref. [6] for (a), © Science China Press 2020. \| https://mc03.manuscriptcentral.com/friction Friction 11(6): 839–864 (2023) 851 solid–liquid interface contact area and interface bonding force, which caused slippage at the solid–liquid interface and reduced the friction near the wall. Choi et al. [148] studied the effect of superhydrophobic coating on the flow field around the propeller. They found that Never Wet superhydrophobic coating on the surface of a 42 mm diameter double-blade propeller reduced the turbulent kinetic energy (TKE) of the propeller’s wake by an average of about 20%, as shown in Figs. 12(b) and 12(c). Compared with the actual ship propeller, the propeller used by Choi et al. is an order of magnitude smaller in size; and the Reynolds number in the experiment is small, which does not conform to the turbulent working condition of the large Reynolds number in the actual situation. Therefore, the effect of superhydrophobic coating on propeller performance under actual operating conditions needs further study. consumption, developing new superlubricity materials, exploring new characterization methods, and revealing the mechanism of material friction damage. It will be of great meaning to the development of new materials, advanced manufacturing, smart transportation, aerospace, and other fields. 4.1 Friction energy consumption of ship propeller With the increasingly severe global warming problem, energy conservation and emission reduction have become more and more important in the shipping industry. According to the statistics [145], the energy consumption of H2O transportation accounts for about 12% of the global transportation energy consumption, of which about 1/3 of the energy is used to drive the propeller (Fig. 12(a)) and overcome the resistance of the ship during the travel. In actual working conditions, the maximum efficiency of the propeller is only about 70%. Even in the most efficient working conditions, the frictional resistance loss at the blade profile is as high as 20%. Therefore, reducing the frictional resistance of the propeller will significantly reduce frictional energy consumption and improve efficiency. By weakening the interaction between the solid– liquid interface to reduce the interfacial frictional resistance, and by using an antifouling coating to solve the biological attachment, the frictional energy consumption of the propeller can be significantly reduced. However, the internal mechanism of superhydrophobic surface affecting the propeller’s efficiency and how to improve the effective service life of the antifouling coating are still unclear. The frictional energy consumption of the propeller comes from two aspects: One is the solid–liquid interface friction at the propeller blade profile, and the other is the biological attachment friction on the surface of the propeller [146]. Solid–liquid interface friction is related to the interaction between the interface. Wang et al. [147] prepared a superhydrophobic surface with a rough structure and a low surface energy coating. This structure could effectively reduce the 4 Friction energy dissipation in high-end equipment The widespread friction and wear phenomena in mechanical systems bring huge energy dissipation and lead to component failures, restricting mechanical system service life and reliability. Here, by analyzing the tribological problems encountered by some key parts of high-end equipment in the fields of navigation, high-speed rail, and precision machining, it illustrates the importance of friction energy dissipation for high-end equipment. The propeller is in long-term contact with seawater during service, and many marine organisms will adhere to its surface. Adhering organisms not only destroy the fluid linearity of the propeller and increase frictional energy consumption, but also produce corrosive extracellular secretions, destroy the surface of the propeller, and increase maintenance costs [149]. The use of marine antifouling coatings is an effective way to inhibit the adhesion of marine organisms. According to Ref. [150], the use of Intersleek900 fluorine-containing modified polymer antifouling coating can weaken the binding force between organisms and solid surfaces, thereby increasing the difficulty of biological attachment, reducing the resistance of the propeller, and saving 6% of fuel consumption. 4.2 Precision machine tool guide Precision computer numerical control (CNC) is the www.Springer.com/journal/40544 \| Friction 852 Friction 11(6): 839–864 (2023) Fig. 12 Research on the TKE of ship propeller wake. (a) Typical ship propeller, (b) smooth surface propeller wake TKE (left) and superhydrophobic surface propeller wake TKE (right), and (c) propeller wake TKE changes at the centerline. ○ and ● denote the smooth surface and superhydrophobic surface, respectively. Reproduced with permission from Ref. [148], © Author(s) 2019. Fig. 12 Research on the TKE of ship propeller wake. (a) Typical ship propeller, (b) smooth surface propeller wake TKE (left) and superhydrophobic surface propeller wake TKE (right), and (c) propeller wake TKE changes at the centerline. ○ and ● denote the smooth surface and superhydrophobic surface, respectively. Reproduced with permission from Ref. [148], © Author(s) 2019. Fig. 13 Regular law of wear and tear of linear motion guideway. (a) Machine linear motion guideway, (b) changing trend of the single wear depth of the edge node of the guideway with the number of wear. Reproduced with permission from Ref. [152], © China Academic Journal Electronic Publishing House 2016. Fig. 13 Regular law of wear and tear of linear motion guideway. (a) Machine linear motion guideway, (b) changing trend of the single wear depth of the edge node of the guideway with the number of wear. Reproduced with permission from Ref. [152], © China Academic Journal Electronic Publishing House 2016. established a wear prediction model, and carried out research on wear reliability prediction. However, these studies can only predict the regular law of wear, and cannot reduce wear from the source and improve precision retention. basic equipment of advanced manufacturing technology. The precision of CNC is inseparable from the linear motion guideway (Fig. 13(a)), and the precision retention is highly related to the friction and wear of the guideway [151]. Li et al. [152] established a calculation formula for the wear depth based on the Archard model, solved it with a discretization method, and proposed a finite element analysis method of the wear of guideway. Zhang and Sun [153] studied the influencing factors of wear from the perspective of randomness in Fig. 13(b). They considered the main factors and micro-random influencing factors, To achieve smooth movement without friction and vibration, the linear guideway currently in ultra- precision photoetching machines is the air-floating guideway. The air-floating guideway is based on gas-dynamic and static pressure. \| https://mc03.manuscriptcentral.com/friction 4.3 High-power gear transmission system in high- speed railway High-speed rail is a “golden business card” of China’s equipment manufacturing. The high-power gear transmission system (Fig. 14(a)), as the core component of high-speed train energy transmission, is not only the key to achieving higher speed, but its reliability and stability directly affect the safe operation of trains. In the gear transmission, friction energy consumption mainly comes from two aspects: One is the contact friction of the surface roughness peak, and the other is the shear friction of the liquid lubricating film. As the gear speed increases, the vibration frequency increases. This leads to the increase of the surface impact strength, the contact frequency of the surface roughness peak, and the solid–solid contact friction. According to traditional lubrication theory, increasing lubricating oil’s viscosity is necessary to increase the 4.2 Precision machine tool guide It has the characteristics of high motion accuracy and low friction and wear, which can maintain manufacturing accuracy for a 853 Friction 11(6): 839–864 (2023) lubricating film’s thickness, thereby reducing the rough peak contact. Still, high-viscosity lubricating oil will increase the shear friction of the lubricating film. Therefore, we hope to reduce the contact of rough peaks while using low-viscosity lubricating oil and the solid–solid contact friction in the gearbox. Solid superlubricity is an effective way to prevent solid–solid contact friction. But under the extreme working conditions of high-speed rail gearboxes, the superlubricity state is difficult to maintain stably. The solid–liquid coupling can improve the stability of the superlubricity state [6], as shown in Fig. 14(b). By introducing the additive of two-dimensional material into the lubricating fluid, it is adsorbed on the surface of the friction pair to form a low-shear interface, reducing the rough peak contact friction under extreme conditions. Meanwhile, the additives are dispersed in the lubricating liquid molecules, which can reduce the interaction between the molecules, thereby forming a shearing lubricating oil film to improve the stability of the superlubricity state. The application of superlubricity in high-speed rail gearboxes will greatly reduce the frictional energy consumption of gear transmission and ensure the safe operation of trains. long time. But, the air-floating guideway has a small bearing capacity, low rigidity, and high manufacturing and installation accuracy requirements, making it difficult to widely application [154]. The emergence of superlubricity technology may be an ideal candidate for traditional linear motion guideways to achieve near-zero friction and wear, in which there is little energy dissipation. By constructing a superlubricity interface on the surface of the guideway, while ensuring the bearing capacity and rigidity, it will greatly reduce the friction and wear between the friction pairs of the guideway, and improve the precision retention and processing reliability. 5 Economic analysis of frictional energy dissipation—A case study of high-speed railway As mentioned above, shipping, railway, machinery manufacturing, and other industries are the important parts of a country’s industry and play essential roles Fig. 14 Two-dimensional material nanosheets improve superlubricity state stability. (a) High-speed rail gear transmission system and (b) nanosheet mechanism. Fig. 14 Two-dimensional material nanosheets improve superlubricity state stability. (a) High-speed rail gear transmission system and (b) nanosheet mechanism. www.Springer.com/journal/40544 \| Friction Friction 11(6): 839–864 (2023) 854 the first place worldwide [158]. In addition to the convenience of travelling for citizens, the achievement of carbon neutralization objectives must be supported by the rapid development of high-speed railways. Compared to the traditional internal combustion engines, the high-speed railway is driven solely by electricity, making it much more environmentally friendly. In the following, we will focus on the electrical energy consumed to overcome friction dissipation and potential economic savings by applying tribology theories and technologies. Some researchers have established models to analyze the energy consumption in cars and trucks [3, 159–161]. According to these methodologies of economic forecasting, Fig. 15 shows the flowchart for CRH. in the national economy, environmental protection, and sustainable development. However, the problem of frictional energy dissipation is becoming more and more prominent in these industries. If frictional energy dissipation can be effectively reduced, it will generate huge economic benefits and promote the green development of human society. This section will explore the impact of tribological applications in the machinery industry and predict potential economic savings accordingly. g g y To better approach the sense of tribology applications in the machinery industry, we must have an idea first of what our Mother Earth is contemporarily facing. The environmental crisis that the world is currently dealing with has been more serious than ever. On the Climate Ambition Summit 2020, the UN secretary- general Antonio Guterres urged all countries to declare climate emergencies. He said, “can anybody still deny that we are facing a dramatic emergency? I urge all others to follow.” [155] Hence, the term carbon- neutral has been brought up as a priority. Multiple countries have updated their longer-term objectives towards zero emissions. China’s action on achieving carbon-neutral objectives is now known as the Fourteenth Five-Year Plan from 2021 to 2025. It specifically and precisely set two goals: decreasing energy consumption per unit of GDP by 13.5% and reducing carbon dioxide emissions by 18% [156]. 5 Economic analysis of frictional energy dissipation—A case study of high-speed railway The machinery industry, in this case, holds indisputable responsibility for achieving these carbon-neutral objectives [157], and so do the tribology applications to the industry. Therefore, the reduction of friction energy dissipation in the machinery industry is necessary for completing the objective of international carbon neutralization. There are two general expected outputs from this evaluation, namely: 1) Estimating the energy dissipation of CRH caused by friction; calculation output with an energy unit of TJ. 2) Estimating the potential economic saving by tribology applications; calculation output with a currency unit of U.S. dollar $. For the evaluation of energy dissipation caused by friction, the process will contain the following steps: 1) Due to the differences in the applied technology, quality of products, working conditions, etc., an “average” equipment of CRH must be defined. Specifically, the equipment is under theoretical conditions and setups based on national data to gather representative conditions. Fig. 15 Methodology flowchart. Next, we take the high-speed rail industry as an example to deeply explore the economic value brought by the application of tribology from the perspective of economics. \| https://mc03.manuscriptcentral.com/friction 5.1 Methodology of economic savings forecasting for China Railway High-speed (CRH) The forecast method for the second objective is shown as follows: 1) Contemporary economic savings: converting the energy dissipation saved by tribological applications into the saved amount of electricity used by the equipment with the most leading lab technology. Then calculate it using the national average price of industrial electricity. 1) Contemporary economic savings: converting the energy dissipation saved by tribological applications into the saved amount of electricity used by the equipment with the most leading lab technology. Then calculate it using the national average price of industrial electricity. Table 2 CRH380B high-speed train parameters [163]. Table 2 CRH380B high-speed train parameters [163]. Type CRH380B Operation speed 350 km/h Composition 8M8T Seating capacity 1,042 persons Length of trainset ~400 m Traction power 18,400 kW Emergency brake distance < 6500 m Length of carbody 24,175 mm Width of carbody 3,265 mm Height of carbody 3,890 mm Height of floor from rail top 1,260 mm Bogie CW400 Axle load 17 t 2) 5-year economic savings: converting the energy dissipation saved by tribological applications into the saved amount of electricity used by the equipment. Then calculate it using the estimated future national average price of industrial electricity. 5.1 Methodology of economic savings forecasting for China Railway High-speed (CRH) CRH is one of China’s most influential transportation infrastructure and machinery industry sectors. As of the end of 2019, the total mileage of domestic high- speed rail reached 35,000 km, which was ranked in Fig. 15 Methodology flowchart. \| https://mc03.manuscriptcentral.com/friction Friction 11(6): 839–864 (2023) 855 Table 1 Type of CRH in leading service counts among every speed level [162]. Speed level (km/h) Type Count 300–350 CRH380B 353 200–250 CRH2A 491 160 CRH6F-A 20 Table 1 Type of CRH in leading service counts among every speed level [162]. 2) The overall energy consumption process and status are analyzed based on the “average” equipment conditions according to investigations and research. Then determine the proportion of friction loss, hence calculating the total friction-related energy dissipation. 2) The overall energy consumption process and status are analyzed based on the “average” equipment conditions according to investigations and research. Then determine the proportion of friction loss, hence calculating the total friction-related energy dissipation. 2) The overall energy consumption process and status are analyzed based on the “average” equipment conditions according to investigations and research. Speed level (km/h) Type Count 300–350 CRH380B 353 200–250 CRH2A 491 160 CRH6F-A 20 Then determine the proportion of friction loss, hence calculating the total friction-related energy dissipation. Then determine the proportion of friction loss, hence calculating the total friction-related energy dissipation. 3) Evaluating the energy dissipation savings by tribology applications. The time span of saving includes the highest level of technologies that can be applied on a large scale contemporarily; the potential level of technologies that may be realized in 5 years. Even though the count for CRH2A is the highest, we decided to use CRH380B as the average equipment for two reasons. First, the total count for 300–350 km/h speed level CRH is 1,809, holding 59.18% of the total CRH in service. On the other hand, the future development of China’s high-speed railways will replace the slower types with higher speed and hence develop better efficiencies. Only by sticking with that direction, we may achieve the carbon-neutral objectives by 2025. The following presents the tech parameters of CRH380B in Table 2. With the previous steps completed, the methodology should develop from the output of the first objective of forecasting to the second, which is the potential economic savings. 5.2 Forecasting potential economic savings on CRH by tribological applications According to the reports [168, 169] and statistics [170], the national annual passenger transport quantity and passenger turnover quantity are concluded in www.Springer.com/journal/40544 \| Friction www.Springer.com/journal/40544 \| Friction Friction 11(6): 839–864 (2023) 856 forecasts are shown as following: CRH380B with leading tribological technology applications—2020 annually Total electricity used to overcome friction: 2,765.25 (106 kWh) Saving proportion compared to CRH380B 2020: 25% Saving in electricity energy: 3,318.30 (103 TJ) Saving in economic value: 142.78 (106 $) CRH380B with average tribological technology applications—2025 annually Total electricity used to overcome friction: 1,685.15 (106 kWh) Saving proportion compared to CRH380B 2020: 55% Saving in electricity energy: 7,300.26 (103 TJ) Saving in economic value: 314.11 (106 $) The proportion of saving above is relatively forecasts are shown as following: CRH380B with leading tribological technology applications—2020 annually Total electricity used to overcome friction: 2,765.25 (106 kWh) Saving proportion compared to CRH380B 2020: 25% Saving in electricity energy: 3,318.30 (103 TJ) Saving in economic value: 142.78 (106 $) CRH380B with average tribological technology applications—2025 annually Total electricity used to overcome friction: 1,685.15 (106 kWh) Saving proportion compared to CRH380B 2020: 55% Saving in electricity energy: 7,300.26 (103 TJ) Saving in economic value: 314.11 (106 $) forecasts are shown as following: Table 3. Based on the two parameters, the average transportation distance can then be calculated. Table 3 Travelling parameters of CRH in 2020. Passenger transport quantity Passenger turnover quantity Average transportation distance 1.43 billion passengers 485.14 billion passenger–km 339.21 km Table 3 Travelling parameters of CRH in 2020. CRH380B with average tribological technology applications—2025 annually CRH380B with average tribological technology applications—2025 annually Thus, the total electricity consumption by CRH380B as average equipment in 2020 can be calculated as Total electricity used to overcome friction: 1,685.15 (106 kWh) Total electricity used to overcome friction: 1,685.15 (106 kWh) Passenger transport quantity 100 electricity consumption per 100 passenger–km × average transportation distance = 1.84×1010 kWh (1) Passenger transport quantity 100 electricity consumption per 100 passenger–km × average transportation distance = 1.84×1010 kWh (1) Saving proportion compared to CRH380B 2020: 55% Saving proportion compared to CRH380B 2020: 55% Saving in electricity energy: 7,300.26 (103 TJ) Saving in economic value: 314.11 (106 $) (1) The proportion of saving above is relatively conservative already. Optimistically, superlubricity applications will reduce the friction resistances ultimately close to zero. 5.2 Forecasting potential economic savings on CRH by tribological applications The first step is to build up average equipment, in this case, a certain type of CRH represented in the entire field. When defining “average”, our priority criterion is to pick an existing type of train instead of building up an ideal but not real equipment. Because the purpose of this economic forecast is to give people with interests to understand the potential realistic effect of applying high-end technology of tribology, we decided that the most representative type of CRH should be the one that is mostly employed and provides the services. According to The World Encyclopedia of High-speed Trains [162], all types of CRH are classified by their speed levels, including 300–350, 200–250, and 160 km/h. A part of the data extracted from the encyclopedia is shown by the following Table 1. The next step is to build up a starting point of its energy consumption rate, hence estimating the annual energy consumption of the CRH system. By conducting research and summarizing multiple reports [164–167], the energy consumption efficiency of CRH380B is estimated to be 3.8 kWh per 100 passenger-km. With energy consumption efficiency, the annual passenger volume with total distance travelled is also required. Total annual energy consumption 13,273.20 TJ  (3) Total annual energy consumption 13,273.20 TJ  (3) The last step is to convert the energy savings into economic ones by adapting the current China’s industrial electricity price of 0.1549 $/kWh, which is also considered stable for the following five years. Noticeably, the potential reduction in the energy used to overcome friction in advanced tribological applications today and future five years are 25% and 55%, respectively [161, 174]. The calculations and Fig. 16 Potential economic savings by advanced tribology applications in CRH by time scale. 5.2 Forecasting potential economic savings on CRH by tribological applications Figure 16 represents the ideal case of long-term saving that may be reached by the development of tribology considering approximate inflations of 7% annually. The immense amount of economic savings with the potential tribological application, dedicated research, and innovations are required for its realization. The world achieving carbon-neutral objectives is not just saving the contemporary human being, and it is our legacy for the following generations, our commitment to the future. And the amount of energy consumption converted is 10 1.8435 10 kWh 3,600,000 J 66,366 TJ    (2) Since the total energy consumption is obtained, the proportion of energy used to overcome friction can be estimated. For the equipment like CRH train, the assumption is that the major running resistance, where most energies are used to overcome aerodynamic and frictional resistances. For CRH, when the train runs at 200 km/h, the aerodynamic resistance accounts for about 70% of the total resistance. When it runs at 486.1 km/h, the aerodynamic resistance exceeds 92% of the total resistance. If it runs at more than 500 km, the proportion becomes more than 95% of the total [171]. Moreover, the proportion of total energy consumption used to overcome aerodynamic resistance is estimated to be 80%, hence leaving 20% to frictional resistance [172]. Therefore, the energy converted to overcome friction is determined [173]: Fig. 16 Potential economic savings by advanced tribology applications in CRH by time scale. \| https://mc03.manuscriptcentral.com/friction 6 Conclusions Therefore, the study of friction energy dissipation should be transformed from mechanics to the perspective of ultrafast phonon and electron energy dissipation. The ultrafast spectroscopy is an effective tool to detect ultrafast energy dynamics. If the ultrafast spectroscopy and friction detection platform are combined, it is expected to realize in-situ real-time detection of ultrafast friction phonon and electron energy dissipation, which provides a powerful means to explore the nature of superlubricity and the origin of friction. Acknowledgements This work is supported by the National Natural Science Foundation of China (Nos. 52075284, 52105195, 51527901, and 11890672) and the Postdoctoral Research Foundation of China (Nos. 2020M680528 and BX2021151). This work is supported by the National Natural Science Foundation of China (Nos. 52075284, 52105195, 51527901, and 11890672) and the Postdoctoral Research Foundation of China (Nos. 2020M680528 and BX2021151). 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. One of our research purposes on friction energy dissipation is to clarify the energy dissipation channels of friction interfaces. By regulating the energy dissipation channels, friction can be reduced or inhibited from the source to realize the active design of superlubricity interfaces. Superlubricity is a disruptive technology with huge application prospects in high-end equipment fields such as precision manufacturing, aviation, aerospace, and new energy vehicles. It provides an important way to solve common problems such as high energy consumption and poor stability of the equipment. Here, by analyzing the tribology problems encountered by some key parts of high-end equipment in the fields of navigation, high-speed rail, and precision machining, it illustrates the importance of friction energy dissipation for high-end equipment. Finally, we analyze the impact of friction consumption reduction on the high-speed rail from an economic point of view. We predict that huge energy losses will To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. 6 Conclusions In summary, we have reviewed the classical microscopic mechanism of friction energy dissipation, including the phonon dissipation model (PT and FKT) \| https://mc03.manuscriptcentral.com/friction Friction 11(6): 839–864 (2023) 857 be saved if superlubricity is applied to the high- speed railway. Therefore, the study of friction energy dissipation is of great significance to high-end equipment, green energy, and economic development, and is a strong support for the world to achieve carbon-neutral. and the electron dissipation model (Coulomb drag). Research about these atomic models mainly focuses on simulation calculations, and some experiments have proved the significance of phonon and electron dissipation in friction. However, due to the complex interaction of the friction interface, these experiments usually study the friction energy dissipation through changes in macroscopic properties. The emergence of non-contact friction avoids various influencing factors compared with direct contact, in which vacuum and low temperature provide a pure experimental environment for studying the electron and phonon dissipation. Furthermore, since the electron and phonon dissipation usually occur in an ultrafast time range (femtoseconds to nanoseconds) at the nanoscale, this has reached the detection limit of existing scientific instruments in the field of tribology. Therefore, the study of friction energy dissipation should be transformed from mechanics to the perspective of ultrafast phonon and electron energy dissipation. The ultrafast spectroscopy is an effective tool to detect ultrafast energy dynamics. If the ultrafast spectroscopy and friction detection platform are combined, it is expected to realize in-situ real-time detection of ultrafast friction phonon and electron energy dissipation, which provides a powerful means to explore the nature of superlubricity and the origin of friction. and the electron dissipation model (Coulomb drag). Research about these atomic models mainly focuses on simulation calculations, and some experiments have proved the significance of phonon and electron dissipation in friction. However, due to the complex interaction of the friction interface, these experiments usually study the friction energy dissipation through changes in macroscopic properties. The emergence of non-contact friction avoids various influencing factors compared with direct contact, in which vacuum and low temperature provide a pure experimental environment for studying the electron and phonon dissipation. 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References Tribol Int 78: 94–114 (2014) [173] Yuan Z W, Wu M L, Tian C, Zhou J J, Chen C. A review on the application of friction models in wheel–rail adhesion calculation. Urban Rail Transit 7(1): 1–11 (2021) [174] Information on https://news.cgtn.com/news/2021-01-27/ China-unveils-Maglev-technology-to-challenge-world-s- fastest-trains-XozVNbOSPe/index.html. [160] Holmberg K, Kivikytö-Reponen P, Härkisaari P, Valtonen K, Erdemir A. Global energy consumption due to friction and wear in the mining industry. Tribol Int 115: 116–139 (2017) Huan LIU. He received B.S. degree in materials forming and control engineering in 2013 from Northwestern Polytechnical University, China. And he received M.S. degree in physics in 2016 and Ph.D. degree in mechanical Huan LIU. He received B.S. degree in materials forming and control engineering in 2013 from Northwestern Polytechnical University, China. And he received M.S. degree in physics in 2016 and Ph.D. degree in mechanical Huan LIU. He received B.S. degree in materials forming and control engineering in 2013 from Northwestern Polytechnical University, China. And he received M.S. degree in physics in 2016 and Ph.D. degree in mechanical engineering in 2020 from Tsinghua University, China. He is currently a post-doctor in the State Key Laboratory of Tribology at Tsinghua University. His research interests include friction energy dissipation of superlubricity and ultrafast electron dynamics in moiré superlattices. www.Springer.com/journal/40544 \| Friction 864 Friction 11(6): 839–864 (2023) of study fall into quantitative econometrics and statistic. During his undergraduate period, he was also actively involved in campus sustainability and related research. Boming YANG. He obtained his bachelor’s degree in 2021 from the University of Illinois, Urbana- Champaign, USA. His major fields at the State Key Laboratory of Tribology, Tsinghua University. His research interest is non-contact friction energy dissipation mechanism of two-dimensional materials. Chong WANG. He obtained his bachelor’s degree in 2020 from Tsinghua University, China. After then, he became a Ph.D. student under the supervisor of Prof. LUO of Prof. LIU at the State Key Laboratory of Tribology, Tsinghua University. Her research interest is the quantum behavior of excitons at two-dimensional materials friction interface. Yishu HAN. She obtained her bachelor’s degree in 2021 from Harbin Institute of Technology, China. After then, she became a Ph.D. student under the supervisor of Prof. LIU at the State Key Laboratory of Tribology, Tsinghua University. Her research interest is the quantum behavior of excitons at two-dimensional materials friction interface. energy dissipation mechanism, photoelectric non- destructive testing technology, composite testing equipment, and flexible material manufacturing equipment. \| https://mc03.manuscriptcentral.com/friction References He has authored over 70 SCI papers, including Advanced Materials, ACS Nano, Nano Letters and other high-impact journals. Dameng LIU. He obtained his Ph.D. degree from University of Cambridge, UK. He is an associate professor and a doctoral supervisor at Tsinghua University, China. In the last couple of years, his main research interests include friction \| https://mc03.manuscriptcentral.com/friction
https://openalex.org/W2048628486	https://journals.plos.org/plosgenetics/article/file?id=10.1371/journal.pgen.1002987&type=printable	English	null	A Mimicking-of-DNA-Methylation-Patterns Pipeline for Overcoming the Restriction Barrier of Bacteria	PLOS genetics	2,012	cc-by	13,246	Abstract 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. Funding: This work was supported by grants from the Chinese Academy of Sciences (KSCX2-EW-J-6) and the Ministry of Science and Technology of China (2008ZX09401-05 and 2010ZX09401-403). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: wenty@im.ac.cn Guoqiang Zhang1, Wenzhao Wang2, Aihua Deng1, Zhaopeng Sun1, Yun Zhang1, Yong Liang1, Yongsheng Che3, Tingyi Wen1* Guoqiang Zhang1, Wenzhao Wang2, Aihua Deng1, Zhaopeng Sun1, Yun Zhang1, Yong Liang1, Yongsheng Che3, Tingyi Wen1* 1 Department of Industrial Microbiology and Biotechnology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China, 2 State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China, 3 Department of Natural Products Chemistry, Beijing Institute of Pharmacology and Toxicology, Beijing, China Abstract Genetic transformation of bacteria harboring multiple Restriction-Modification (R-M) systems is often difficult using conventional methods. Here, we describe a mimicking-of-DNA-methylation-patterns (MoDMP) pipeline to address this problem in three difficult-to-transform bacterial strains. Twenty-four putative DNA methyltransferases (MTases) from these difficult-to-transform strains were cloned and expressed in an Escherichia coli strain lacking all of the known R-M systems and orphan MTases. Thirteen of these MTases exhibited DNA modification activity in Southwestern dot blot or Liquid Chromatography–Mass Spectrometry (LC–MS) assays. The active MTase genes were assembled into three operons using the Saccharomyces cerevisiae DNA assembler and were co-expressed in the E. coli strain lacking known R-M systems and orphan MTases. Thereafter, results from the dot blot and restriction enzyme digestion assays indicated that the DNA methylation patterns of the difficult-to-transform strains are mimicked in these E. coli hosts. The transformation of the Gram-positive Bacillus amyloliquefaciens TA208 and B. cereus ATCC 10987 strains with the shuttle plasmids prepared from MoDMP hosts showed increased efficiencies (up to four orders of magnitude) compared to those using the plasmids prepared from the E. coli strain lacking known R-M systems and orphan MTases or its parental strain. Additionally, the gene coding for uracil phosphoribosyltransferase (upp) was directly inactivated using non-replicative plasmids prepared from the MoDMP host in B. amyloliquefaciens TA208. Moreover, the Gram-negative chemoautotrophic Nitrobacter hamburgensis strain X14 was transformed and expressed Green Fluorescent Protein (GFP). Finally, the sequence specificities of active MTases were identified by restriction enzyme digestion, making the MoDMP system potentially useful for other strains. The effectiveness of the MoDMP pipeline in different bacterial groups suggests a universal potential. This pipeline could facilitate the functional genomics of the strains that are difficult to transform. Citation: Zhang G, Wang W, Deng A, Sun Z, Zhang Y, et al. (2012) A Mimicking-of-DNA-Methylation-Patterns Pipeline for Overcoming the Restriction Barrier of Bacteria. PLoS Genet 8(9): e1002987. doi:10.1371/journal.pgen.1002987 Editor: Josep Casadesu´s Universidad de Sevilla Spain Citation: Zhang G, Wang W, Deng A, Sun Z, Zhang Y, et al. (2012) A Mimicking-of-DNA-Methylation-Patterns Pipeline for Overcoming the Restriction Barrier of Bacteria. PLoS Genet 8(9): e1002987. doi:10.1371/journal.pgen.1002987 Received July 20, 2012; Accepted August 10, 2012; Published September 27, 2012 Copyright: 2012 Zhang et al. This is an open-access article distributed under the terms of the Creative Commons Attributi unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. ang et al. Cloning and characterization of putative MTases g p MTases modify DNA by adding a methyl group to the individual bases, thereby preventing DNA cleavage by the corresponding REases. In the MoDMP procedure, MTases from the difficult-to-transform bacterial strains were used to protect DNA from being degraded by the REases. The genes of 24 putative MTases including two belonging to Type I R-M systems, 19 belonging to Type II R-M systems, two belonging to Type III R-M systems, and one orphan MTase were cloned from the genomes of the three difficult-to-transform strains into the pBAD43 vector (Table 1). been performed in B. cereus ATCC 10987 due to its resistance to genetic manipulation. Transformation of B. cereus ATCC 10987 has been performed with DNA prepared from Bacillus subtilis with low efficiency [13,14], and four REases have recently been characterized [15]. Bacillus amyloliquefaciens TA208 is an industrial guanosine-producing strain [16] and has been reported to be transformed at low efficiencies with plasmids prepared from Escherichia coli [17]. been performed in B. cereus ATCC 10987 due to its resistance to genetic manipulation. Transformation of B. cereus ATCC 10987 has been performed with DNA prepared from Bacillus subtilis with low efficiency [13,14], and four REases have recently been characterized [15]. Bacillus amyloliquefaciens TA208 is an industrial guanosine-producing strain [16] and has been reported to be transformed at low efficiencies with plasmids prepared from Escherichia coli [17]. To date, three types of methyl-transferring activity have been described for bacterial DNA MTases, namely N6-methyladenine (m6A), N4-methylcytosine (m4C) and 5-methylcytosine (m5C) modifications. Dot blot assays were conducted to detect the modified bases in the total genomic DNA of the E. coli EC135 strains expressing individual MTase using antibodies against m6A, m4C and m5C. In total, 13 of the putative MTase genes exhibited methyl transfer activity to DNA (Figure 1), and the bases they modified are summarized in Table 1. The spots of the dot blots were also scanned and quantified, and the relative intensity of each spot is shown in Figure S2. Here, we describe a mimicking-of-DNA-methylation-patterns (MoDMP) pipeline. An E. coli strain lacking all of the known six characterized R-M systems and orphan MTases was generated to prevent unintentional modification of propagated plasmids or cleavage of DNA with foreign methylation patterns. After expressing multiple active MTases from the target bacteria in the E. coli strain lacking known R-M systems and orphan MTases, the DNA methylation patterns of E. Introduction coli mutant lacking all of the six characterized R-M systems and orphan MTases genes, namely strain EC135, was generated in the E. coli TOP10 background by deleting the dam and dcm genes. A wild-type recA allele was introduced into the strain prior to dam inactivation to counteract the inviability of the dam recA double mutant strain [20]. The construction of the E. coli EC135 strain is explained in detail in the Supporting Information Methods (Text S1), and validation of the strain is described in the Supporting Information Results (Text S1 and Figure S1). REases that restrict methylated DNA (Mrr, McrA and MrcBC) should be inactivated in the MoDMP host. To date, three E. coli strains that do not methylate DNA or restrict DNA with foreign methylation patterns have been described (E. coli DB24 [18], E. coli HST04 from Clontech and E. coli JTU007 [19]). In this study, an E. coli mutant lacking all of the six characterized R-M systems and orphan MTases genes, namely strain EC135, was generated in the E. coli TOP10 background by deleting the dam and dcm genes. A wild-type recA allele was introduced into the strain prior to dam inactivation to counteract the inviability of the dam recA double mutant strain [20]. The construction of the E. coli EC135 strain is explained in detail in the Supporting Information Methods (Text S1), and validation of the strain is described in the Supporting Information Results (Text S1 and Figure S1). Author Summary Cloning and characterization of putative MTases coli were altered to reflect the patterns of the target bacteria. Plasmids prepared from these hosts escaped the host REases, and genetic manipulation could be readily achieved. The pipeline was shown to be effective in all of the three aforementioned strains which are difficult to transform using conventional methods. We report the first genetic transfor- mation of Nitrobacter, the improvement of transformation efficiency by exogenous plasmids in B. cereus ATCC 10987 and B. amyloliquefaciens TA208 using the MoDMP pipeline, and direct mutagenesis using non-replicative plasmids in B. amyloliquefaciens TA208. The MoDMP pipeline may be readily adapted to bacteria carrying multiple R-M systems. The hybridization signals of BCE_0392, Nham_0582, Nham_0803 and Nham_3225 were weak in the dot blot experiments. To confirm their activity, the total DNA of the E. coli EC135 strains expressing these four MTases individually were digested to deoxynucleosides, and Liquid Chromatography-Mass Spectrometry (LC-MS) assays were performed to detect N6- methyl-29-deoxyadenosine (m6dA) in the DNA. In High Perfor- mance Liquid Chromatography-Quadrupole Time-of-Flight/ Mass Spectrometry (HPLC-QTOF/MS) analysis, m6dA (m/z 266.12) was readily detected in the digested DNA of the BCE_0392-, Nham_0582- and Nham_3225-expressing strains in the MS spectrum at the corresponding retention time of standard m6dA, validating that BCE_0392, Nham_0582, and Nham_3225 displayed DNA m6A modification activity in E. coli (Figure 2). Xu et al. has reported that MTase activity was not detected for in vivo translated BCE_0392 protein using [H3]AdoMet and phage l DNA or pXbaI plasmid DNA as substrate [15]. This might either be caused by the mis-folding of in vivo translated BCE_0392 protein or by the absence of BCE_0392 recognition sites from the substrate DNA they used. Author Summary Approximately 95% of the genome-sequenced bacteria harbor Restriction-Modification (R-M) systems. R-M sys- tems usually occur in pairs, i.e., DNA methyltransferases (MTases) and restriction endonucleases (REases). REases can degrade invading DNA to protect the cell from infection by phages. This protecting machinery has also become the barrier for experimental genetic manipulation, because the newly introduced DNA would be degraded by the REases of the transformed bacteria. In this study we have developed a pipeline to protect DNA by methylation from cleavage by host REases. Multiple DNA MTases were cloned from three difficult-to-transform bacterial strains and co-expressed in an E. coli strain lacking all of the known endogenous R-M systems and orphan MTases. Thus, the DNA methylation patterns of these strains have become similar to that of the difficult-to-transform strains. Ultimately, the DNA prepared from these E. coli strains can overcome the R-M barrier of the bacterial strains that are difficult to transform and achieve genetic manipulation. The effectiveness of this pipeline in different bacterial groups suggests a universal potential. This pipeline could facilitate functional genomics of bacterial strains that are difficult to transform. Introduction their subunit composition, cleavage sites, sequence specificity and cofactor requirement [6]. Type I, II and III REases cleave unmethylated DNA at specific sites, and Type IV cut methylated DNA with foreign patterns [6]. R-M systems are believed to act as defenses to protect the prokaryotic cells against invading DNA; exogenous DNA with foreign methylation patterns are recognized and rapidly degraded [7]. Inevitably, this defensive machinery hinders the experimental genetic manipulation of many bacteria species. Moreover, genetic modification becomes even more difficult when the targeted bacteria carry multiple R-M systems. Experimental genetic manipulation has been an essential tool for gaining insight into the significance of bacterial metabolism, physiology and pathogenesis [1,2] and has been instrumental in developing microbial biotechnology [3]. To date, only a limited proportion of the laboratory culturable bacterial species are amenable to genetic manipulation. Among these manipulation- friendly species, many strains are refractory to transformation by exogenous DNA. The currently available laboratory model species satisfied the research need for genetic uniformity, but the handicap in genetic manipulation is a challenge when exploring the unique traits of these non-model species/strains [4]. The Nitrobacter hamburgensis X14 strain oxidizes nitrite to conserve energy and is commonly used in nitrification research [8]. Although the strain was isolated more than 100 years ago [9], limited research on this strain has been published due to the lack of genetic manipulation tools. Genomic sequencing has revealed eleven sets of R-M genes in N. hamburgensis X14 [10]. Bacillus cereus ATCC 10987 is a non-lethal strain in the same genetic subgroup as B. anthracis [11]. Although genetic manipulation has been routine in many other B. cereus strains [12], limited research has Restriction-Modification (R-M) systems are composed of restriction enzymes (REases) and DNA methyltransferases (MTases). These systems are widespread in both bacteria and archaea. Approximately 95% of the genome-sequenced bacteria harbor R-M systems, and 33% carry more than four REases [5]. R-M systems have been classified into four groups depending on September 2012 \| Volume 8 \| Issue 9 \| e1002987 1 PLOS Genetics \| www.plosgenetics.org A Pipeline for Overcoming Restriction Barrier REases that restrict methylated DNA (Mrr, McrA and MrcBC) should be inactivated in the MoDMP host. To date, three E. coli strains that do not methylate DNA or restrict DNA with foreign methylation patterns have been described (E. coli DB24 [18], E. coli HST04 from Clontech and E. coli JTU007 [19]). In this study, an E. sequence specificity analysis has revealed that Nham_0569 modified GATC sequences (see below). approaches could be useful in detecting the possible modified nucleoside conferred by Nham_0803 [21]. It is noteworthy that the E. coli strain EC135 expressing the Nham_0569 MTase grows much slower than the control strain or strains expressing other MTases (Nham_0803 and Nham_3225), and the final biomass of strain EC135 carrying Nham_0569 was about 60% of that for the control strains (Figure S3). This growth retardation in E. coli may be attributed to the toxicity of the Nham_0569 MTase. The E. coli EC135 strain lacks methylation- dependent REases activity, which will cleave its own DNA when foreign methylation patterns are detected, leading to cell death; however, the modification of m6A by the Nham_0569 MTase may occur on sequences overlapping with Dam sites in E. coli, which participates in DNA mismatch repair and replication initiation. Consequently, the premature and untimely methylation of DNA may interfere with strain proliferation [22]. Subsequent Generation and validation of the E. coli strain lacking known R-M systems and orphan MTases R = A or G, Y = C or T, N = A or T or G or C, D = A or G or T, H = A or C or T, W = A or T. P, partial modification. ND, not detected. doi:10.1371/journal.pgen.1002987.t001 sequence specificity analysis has revealed that Nham_0569 modified GATC sequences (see below). Generation and validation of the E. coli strain lacking known R-M systems and orphan MTases Although m6dA was not detected for Nham_0803 in the HPLC-QTOF/MS analysis (Figure 2), the MTase activity of Nham_0803 could not be ruled out, since the DNA of the Nham_0803-expressing strain displayed slight but noticeable signal increase compared with the E. coli EC135 strain harboring empty vector (Figure S2). Other more sensitive and targeted MS To avoid the unintentional activation of the Type IV R-M systems in the target bacteria, plasmids that are to be used for genetic transformation should be prepared from an E. coli host that does not methylate DNA (dam- dcm- hsdRMS-). Moreover, the expression of MTases in E. coli would induce foreign patterns of modification on the E. coli chromosomal DNA; therefore, the September 2012 \| Volume 8 \| Issue 9 \| e1002987 September 2012 \| Volume 8 \| Issue 9 \| e1002987 PLOS Genetics \| www.plosgenetics.org PLOS Genetics \| www.plosgenetics.org 2 A Pipeline for Overcoming Restriction Barrier Table 1. The MTases involved in this study and their characteristics. MTase Type Modification base Sequence specificity B. amyloliquefaciens TA208 BAMTA208_6525 II m4C RGATCY, TGATCA (P) BAMTA208_6715 orphan m5C GGCC, GCNGC, GDGCHC BAMTA208_14440 II ND BAMTA208_19835 II m5C GCWGC BAMTA208_16660 II m4C GGATCC B. cereus ATCC 10987 BCE_0841-CE_0842 I ND BCE_0839-CE_0842 I ND BCE_0365 II m5C GCWGC BCE_0392 II m6Aa BCE_0393 II m5C GCWGC, GGCC, CCGG, GGNNCC, GCGCGC, GGWCC, CCWGG (P) BCE_4605 II m5C GGWCC BCE_5606 II m4C ACGGC(N)12/14 BCE_5607 II m4C ACGGC(N)12/14 BCE_1018 III ND N. hamburgensis X14 Nham_0569 II m6A GATC (P), CAGCTG (P), AATATT (P), ACTAGT (P), CAATTG (P), CATG (P), ATTAAT (P), GANTC (P) Nham_0582 III m6Aa Nham_0803 II NDb Nham_0842 II ND Nham_1185 II ND Nham_1353 II ND Nham_2515 II ND Nham_3225 II m6Aa GANTC Nham_3845 II ND Nham_4499 II ND aWeak signal in dot blot assays and verified by HPLC-QTOF/MS analysis; bWeak m6A signal in dot blot assays and not detected in HPLC-QTOF/MS analysis. Putative modification bases of the MTases have been underlined. R = A or G, Y = C or T, N = A or T or G or C, D = A or G or T, H = A or C or T, W = A or T. P, partial modification. ND, not detected. aWeak signal in dot blot assays and verified by HPLC-QTOF/MS analysis; bWeak m6A signal in dot blot assays and not detected in HPLC-QTOF/MS analysis. Putative modification bases of the MTases have been underlined. Co-expression of multiple active MTases coli EC135 DNA in vivo methylated by M.BamHI and 150 ng of E. coli EC135 DNA in vivo methylated by M.AluI (arranged from top to bottom), were used as controls in each ‘‘Control+’’ column for the m6A, m4C, and m5C experiments. All experiments were repeated at least three times, and representative results are shown. doi:10 1371/journal pgen 1002987 g001 The B. amyloliquefaciens TA208 strain could not be transformed with plasmids prepared from E. coli TOP10 cells but could be transformed with plasmids from the E. coli EC135 strain with low efficiency; this result indicates that a methylation-dependent Type IV R-M system may exist in B. amyloliquefaciens TA208, although its coding gene was not found during annotation of the genome sequence [16]. Hence it may also be that the plasmids methylated at the Dam and Dcm sites would not be inherited in B. amyloliquefaciens TA208, e.g., methylated replication origin would not be bound by the replication protein. The MoDMP protocol increased the transformation efficiencies of all the plasmids tested in B. amyloliquefaciens TA208. The pMK4 plasmid from MoDMP hosts showed the highest transformation efficiency (36106 CFU/ mg DNA), representing a 104-fold increase compared to that of the plasmids from the E. coli EC135 strain. The MoDMP procedure also enabled two previously untransformable plasmids, pAD123 and pDG148StuI, to be transformed at an efficiency of 16105 CFU/mg DNA (Figure 5A). DNA from the E. coli EC135 strain expressing multiple MTases was also tested by dot blot assay, with the DNA of the parent strains as the positive controls (Figure 4). DNA from the co- expression strains exhibited multiple methylation signals, indicat- ing the alteration of the DNA methylation patterns in E. coli. It is worth noting that the m4C and m5C signals in B. amyloliquefaciens strain TA208, the m5C signal in B. cereus strain ATCC 10987 and the m4C signal in N. hamburgensis strain X14 were much weaker when compared with their corresponding MTase over-expressing E. coli strain. This signal weakness could be attributed to the different number of MTase target sequences between the genomic sequences of E. coli and the parent strains or to the fact that the B. amyloliquefaciens TA208 strain is an adenine auxotroph, which limits the availability of S-adenosylmethionine (AdoMet). However, pMK4 plasmid DNA prepared from the TA208 strain is resistant to BamHI digestion, which is a homolog to the restriction subunit of the BAMTA208_16650-BAMTA208_16660 systems (see be- low). Co-expression of multiple active MTases To mimic the DNA methylation patterns of the strains that are difficult to transform, we co-expressed the active MTases from each strain. By taking advantage of the high rates of recombination in Saccharomyces cerevisiae, MTase genes, with optimized ribosome binding site (RBS) for expression in E. coli, were inserted into the pWYE724 backbone to form three operons. The diagrams of the pMoDMP plasmids are shown in Figure 3. The insertion of the MTase genes was verified by multiple methods, including PCR analysis of plasmids, restriction digestion (Figure S4) and DNA sequencing. The protocol for DNA assembly in S. cerevisiae is very powerful, and up to eight MTase genes from Nitrosococcus oceani ATCC 19707 could be readily assembled in our lab (Zhang et al., unpublished). September 2012 \| Volume 8 \| Issue 9 \| e1002987 PLOS Genetics \| www.plosgenetics.org PLOS Genetics \| www.plosgenetics.org 3 A Pipeline for Overcoming Restriction Barrier Figure 1. Dot blot assay for individual MTase activity. Serially diluted DNA (450 ng–150 ng) was used to test the in vivo methylation by MTases. (A) DNA methylated by the MTases from B. amyloliquefaciens TA208 and B. cereus ATCC 10987. (B) DNA methylated by the MTases from N. hamburgensis X14. Antibodies against m6A, m4C, and m5C were used in the upper, middle, and lower panels, respectively. DNA from the E. coli EC135 strain harboring pBAD43 was used as negative control. The identical DNA, 150 ng DNA of the E. coli TOP10 strain, 150 ng of E. coli EC135 DNA in vivo methylated by M.BamHI and 150 ng of E. coli EC135 DNA in vivo methylated by M.AluI (arranged from top to bottom), were used as controls in each ‘‘Control+’’ column for the m6A, m4C, and m5C experiments. All experiments were repeated at least three times, and representative results are shown. doi:10.1371/journal.pgen.1002987.g001 Figure 1. Dot blot assay for individual MTase activity. Serially diluted DNA (450 ng–150 ng) was used to test the in vivo methylation by MTases. (A) DNA methylated by the MTases from B. amyloliquefaciens TA208 and B. cereus ATCC 10987. (B) DNA methylated by the MTases from N. hamburgensis X14. Antibodies against m6A, m4C, and m5C were used in the upper, middle, and lower panels, respectively. DNA from the E. coli EC135 strain harboring pBAD43 was used as negative control. The identical DNA, 150 ng DNA of the E. coli TOP10 strain, 150 ng of E. Co-expression of multiple active MTases Thus, regulational expression of the R-M systems could also explain the weak blot signals in the parent strains; Hegna et al. has reported that the R-M system is activated when B. cereus is grown in the presence of exogenous DNA [23]. g ( g ) For the B. cereus ATCC 10987 strain, the MoDMP pipeline increased the transformation efficiency of the pMK4 plasmid to 26107 CFU/mg DNA and increased the transformation efficiency of pMK3 by 103 fold compared to those from strains E. coli TOP10 or EC135 (Figure 5B). The plasmids prepared from E. coli TOP10 and EC135 strains showed similar transformation efficiencies, indicating that the putative Type IV R-M systems (BCE_1016 and BCE_2317) in B. cereus ATCC 10987 may be inactive. These results were the same as those obtained in the B. cereus ATCC 14579 strain, which could be transformed by methylated DNA (DNA from non-dam dcm mutant strains) [24], though some researchers prefer to use unmethylated DNA [25]. September 2012 \| Volume 8 \| Issue 9 \| e1002987 A Pipeline for Overcoming Restriction Barrier Figure 2. Analysis of m6dA in the DNA of MTases-expressing E. coli strains using HPLC-QTOF/MS. TIC plots represent the Total Ion Chromatograms of eluted components in the digested DNA samples, and MS plots represent the Mass Spectrum at the corresponding m6dA retention time. In the STD panel, 1.5 mg of dC (deoxycytidine), dA (deoxyadenosine), dG (deoxyguanosine), dT (deoxythymidine) and m6dA (N6- methyl-29-deoxyadenosine) nucleoside standards were analyzed. The retention time of standard m6dA was 7.4 min, and the m/z was 266.12. In other panels, digested genomic DNA was used; the ion chromatogram was extracted at the retention time of m6dA and shown as MS plots. doi:10.1371/journal.pgen.1002987.g002 with chromosome of B. amyloliquefaciens TA208 at the upp locus with a low rate (1026) because it lacks a replication origin for Bacillus (Figure 6A). BS043 was obtained and PCR and sequencing analyses revealed the successful replacement of the upp gene with the chloramphenicol resistance gene in this strain (Figure 6B). Uracil phosphoribosyltransferase converts 5-fluorouracil (5-FU) to 5-fluoro-UMP, which is ultimately metabolized to the toxic compound 5-fluoro-dUMP capable of inhibiting the activity of The MoDMP pipeline increased the plasmid transformation efficiency of Bacillus To determine the efficacy of the MoDMP pipeline, various shuttle plasmids carrying divergent replicons and conferring different antibiotic resistance were used to transform B. amyloli- quefaciens TA208 and B. cereus ATCC 10987. Prior to transforming Bacillus, the shuttle plasmids were methylated in vivo when transformed into the E. coli EC135 strain harboring the pMoDMP plasmids. B. amyloliquefaciens TA208 and B. cereus ATCC 10987 were transformed by these plasmids, and the transformation efficiencies were calculated. The high transformation efficiency achieved with the MoDMP method in both Bacillus strains would allow for the direct inactivation of genes using non-replicative integration plasmids. September 2012 \| Volume 8 \| Issue 9 \| e1002987 PLOS Genetics \| www.plosgenetics.org 4 A Pipeline for Overcoming Restriction Barrier PLOS Genetics \| www.plosgenetics.org 5 September 2012 \| Volume PLOS Genetics \| www.plosgenetics.org 5 September 2012 \| Volume 8 \| Issue 9 \| e1002987 September 2012 \| Volume 8 \| Issue 9 \| e1002987 September 2012 \| Volume 8 \| Issue 9 \| e1002987 PLOS Genetics \| www.plosgenetics.org PLOS Genetics \| www.plosgenetics.org PLOS Genetics \| www.plosgenetics.org Specificity identification for the MTases and the DNA methylation patterns comparison between MoDMP hosts and difficult-to-transform bacteria To make the MTase expression vectors more useful, the modification sequences of MTases were determined when ex- pressed individually or co-expressed. As shown in Figure S6A, BAMTA208_6525 protected plasmid from cleavage by BamHI (GGATCC), BglII (AGATCT), and partially from BclI (TGATCA), indicating that BAMTA208_6525 modifies RGATCY and partial TGATCA sequences. BAMTA208_6715 protected pMK4 from cleavage by HaeIII (GGCC), Fnu4HI (GCNGC) and Bsp1286I (GDGCHC), and BAMTA208_19835 and BAMTA208_16660 protect pMK4 from TseI (GCWGC) and BamHI (GGATCC) cleavage, respectively. When co-expressed, the four active MTases from B. amyloliquefaciens TA208 could protect the plasmids from cleavage by all of the REases tested in the individual expression experiments. However, DNA from the B. amyloliquefaciens TA208 strain was only resistant to BamHI cleavage and partially resistant to Fnu4HI and TseI cleavage (Figure S6B), indicating that the expression of BAMTA208_16660 in the native strain was complete, whereas those of BAMTA208_6715 and BAMTA208_19835 were incomplete, and BAMTA208_6525 was not expressed. Figure 4. Dot blot assay for co-expression of multiple MTases. Serial dilutions of DNA (450 ng–50 ng) from the E. coli EC135 strain harboring pM.Bam, pM.Bce or pM.Nham were tested, and the corresponding DNA from the native host of the MTases was used as positive controls. DNA of the E. coli EC135 strain harboring pWYE724 was used as a negative control. Antibodies against m6A, m4C and m5C were used in the upper, middle, and lower panels, respectively. All experiments were repeated at least three times, and representative results are shown. doi:10.1371/journal.pgen.1002987.g004 For the B. cereus ATCC 10987 strain, BCE_0393 could protect plasmid from cleavage by at least 12 REases, i.e., Fnu4HI (partial), TseI, BbvI (GCAGC), HaeIII, EaeI (YGGCCR), HpaII (CCGG), MspI (CCGG, partial), NlaIV (GGNNCC), BssHII (GCGCGC), HhaI (GCGC, partial), AvaII (GGWCC) and PspGI (CCWGG, partial), and the modification sequences of BCE_0393 were concluded as GCWGC, GGCC, CCGG, GGNNCC, GCGCGC, GGWCC and CCWGG (partial). BCE_0365 protected DNA from cleavage by TseI and BbvI, indicating that it modifies GCWGC sequence, BCE_4605 protect DNA from cleavage by AvaII via modification of GGWCC sequence, and BCE_5606 and BCE_5607 both protect DNA from cleavage by BceAI [ACGGC(N)12/14] (Figure S7A). These results are consistent with the reports of Xu et al. [15], except for that ‘‘GGWCC’’ was added to the modification sequences of BCE_0393 in this study. The multi-specificity nature of the prophage MTase BCE_0393 and its sequence overlapping with other MTases from B. cereus ATCC thymidylate synthetase. A Pipeline for Overcoming Restriction Barrier Figure 4. Dot blot assay for co-expression of multiple MTases. Serial dilutions of DNA (450 ng–50 ng) from the E. coli EC135 strain harboring pM.Bam, pM.Bce or pM.Nham were tested, and the corresponding DNA from the native host of the MTases was used as positive controls. DNA of the E. coli EC135 strain harboring pWYE724 was used as a negative control. Antibodies against m6A, m4C and m5C were used in the upper, middle, and lower panels, respectively. All experiments were repeated at least three times, and representative results are shown. doi:10.1371/journal.pgen.1002987.g004 reported [27]. In this study, the N. hamburgensis X14 strain was transformed with plasmids carrying the Green Fluorescent Protein (GFP) encoding gene gfpmut3a using the MoDMP procedure. Total genomic DNA was extracted from 10 mL of the transformed bacteria cells. The plasmid was rescued to E. coli TOP10 cells, and subsequent plasmid preparation (Figure S5A) and restriction digestion with SalI and PstI (Figure S5B) verified the existence of pWYE561 in the transformed bacterial cell lines. During the subculture process, the bacterial cell lines were monitored for contamination by microscopy and culturing on LB plates at 30uC, and no contamination was observed. Green fluorescent signals were observed in the cytoplasm of Nitrobacter, thereby revealing the successful transformation of Nitrobacter (Figure 7A). The culture may contain multiclonal cell lines because the transformants were enriched twice through successive sub- inoculation of the transformation cell mixture in liquid culture (see Materials and Methods for details). Using flow cytometry, the ratio of fluorescent cells was determined to be 50.37% (Figure 7B), demonstrating that 50.37% of the cells were positive transfor- mants. Clonal cell lines could be obtained by streaking the transformant-enriched culture on nylon membranes placed on solid medium and periodically transferred to fresh plates, as described by Sayavedra-Soto et al. in Nitrosomonas europaea [28]. Specificity identification for the MTases and the DNA methylation patterns comparison between MoDMP hosts and difficult-to-transform bacteria The upp/5-FU module has been widely used in many bacterial species for deletion of genes without introducing antibiotic resistance markers [26]. In contrast with the B. amyloliquefaciens TA208 strain, the BS043 strain could grow on minimal medium (MM) supplemented with 5-FU (Figure 6C). These findings suggest that the MoDMP system elevated transfor- mation efficiencies of exogenous plasmid to enable direct gene inactivation, and the upp gene could be used as a counter-selection marker for the in-frame deletion of genes in B. amyloliquefaciens. Gene inactivation using MoDMP integration plasmids in B. amyloliquefaciens TA208 with chromosome of B. amyloliquefaciens TA208 at the upp locus with a low rate (1026) because it lacks a replication origin for Bacillus (Figure 6A). BS043 was obtained and PCR and sequencing analyses revealed the successful replacement of the upp gene with the chloramphenicol resistance gene in this strain (Figure 6B). Uracil phosphoribosyltransferase converts 5-fluorouracil (5-FU) to 5-fluoro-UMP, which is ultimately metabolized to the toxic compound 5-fluoro-dUMP capable of inhibiting the activity of To further validate the efficacy of the MoDMP procedure, the gene coding for uracil phosphoribosyltransferase (upp) in B. amyloliquefaciens TA208 was selected for inactivation using non- replicative integration plasmids. The B. amyloliquefaciens TA208 strain was transformed with pWYE748 plasmids that had been through the MoDMP host. The pWYE748 plasmid recombines Figure 3. Schematic diagrams of pMoDMP plasmids. S. cerevisiae replication and screening elements are marked in purple; elements for replication, screening and protein expression in E. coli are marked in blue, and MTase genes are marked in red. pM.Bam, pM.Bce and pM.Nham carry MTase genes from B. amyloliquefaciens TA208, B. cereus ATCC 10987 and N. hamburgensis X14, respectively. doi:10.1371/journal.pgen.1002987.g003 Figure 3. Schematic diagrams of pMoDMP plasmids. S. cerevisiae replication and screening elements are marked in purple; elements for replication, screening and protein expression in E. coli are marked in blue, and MTase genes are marked in red. pM.Bam, pM.Bce and pM.Nham carry MTase genes from B. amyloliquefaciens TA208, B. cereus ATCC 10987 and N. hamburgensis X14, respectively. doi:10.1371/journal.pgen.1002987.g003 September 2012 \| Volume 8 \| Issue 9 \| e1002987 September 2012 \| Volume 8 \| Issue 9 \| e1002987 PLOS Genetics \| www.plosgenetics.org 6 A Pipeline for Overcoming Restriction Barrier September 2012 \| Volume 8 \| Issue 9 \| e1002987 Genetic transformation of Nitrobacter using the MoDMP pipeline N. hamburgensis X14 harbors 11 putative R-M systems, and successful genetic transformation of this strain has not been PLOS Genetics \| www.plosgenetics.org PLOS Genetics \| www.plosgenetics.org September 2012 \| Volume 8 \| Issue 9 \| e1002987 7 A Pipeline for Overcoming Restriction Barrier Figure 5. Transformation efficiency of B. amyloliquefaciens TA208 and B. cereus ATCC 10987 with various shuttle plasmids. (A) Transformation efficiency of the B. amyloliquefaciens TA208 strain with various shuttle plasmids prepared from the E. coli TOP10, EC135 and the EC135 harboring pM.Bam. (B) Transformation efficiency of the B. cereus ATCC 10987 strain with various shuttle plasmids prepared from the E. coli TOP10, EC135 and the EC135 harboring pM.Bce. Transformation efficiencies shown are averages of at least three replicates 6 SD. * Not Detected. doi:10.1371/journal.pgen.1002987.g005 Figure 5. Transformation efficiency of B. amyloliquefaciens TA208 and B. cereus ATCC 10987 with various shuttle plasmids. (A) Transformation efficiency of the B. amyloliquefaciens TA208 strain with various shuttle plasmids prepared from the E. coli TOP10, EC135 and the EC135 harboring pM.Bam. (B) Transformation efficiency of the B. cereus ATCC 10987 strain with various shuttle plasmids prepared from the E. coli TOP10, EC135 and the EC135 harboring pM.Bce. Transformation efficiencies shown are averages of at least three replicates 6 SD. * Not Detected. doi:10.1371/journal.pgen.1002987.g005 10987 indicated that it plays a major role in the MoDMP pipeline of this strain. the recently characterized non-specific DNA adenine MTase [30]. Nham_3225 protected DNA from HinfI and TfiI cleavage by modifying GANTC sequence (Figure S8A). The pMK4 plasmids prepared from the E. coli EC135 strains expressing Nham_0582 and Nham_0803 were not resistant to the cleavage by DpnII, EcoRI, DraI, PvuII, SspI, HinfI, HindIII, BspHI, BamHI, SpeI, KpnI, SacI or ApaLI, and the specificity of Nham_0582 and Nham_0803 was not identified. The pMK4 plasmids prepared from the E. coli strain expressing BCE_0392 was challenged with various REases which might be sensitive to m6A modification, including AvaII, BamHI, BbvI, BceAI, BglII, BsiEI, Bsp1286I, BspDI, BstNI, BspHI, DpnII, EaeI, EcoRI, Fnu4HI, HincII, HindIII, HpaII, HinfI, NlaIV, PstI, PshAI, PspGI, SalI, ScrFI, SwaI, SpeI, TaqI and TseI, but resistance to cleavage was not observed. Therefore BCE_0392 might modify sequences that are not recognized by these REase, and new techniques like single-molecule DNA sequencing other than restriction analysis using commercialized REases should be useful in identifying the sequence specificity of BCE_0392 [29]. Genetic transformation of Nitrobacter using the MoDMP pipeline DNA nicking-associated concatenation activity was also detected for BCE_0392 in vivo [15], suggesting that this ParB-Methyltrans- ferase might participate in phage DNA replication or phage packaging, since BCE_0392 was located in a prophage region in the chromosome of the B. cereus ATCC 10987 strain [11]. The four active MTases from N. hamburgensis X14 provided DNA partial protection from cleavage by DpnI, DpnII, PvuII, SspI, SpeI, MfeI, NlaIII, AseI, HinfI and TfiI when co-expressed, and the genomic DNA of the N. hamburgensis X14 strain was sensitive to DpnII digestion, partially resistant to DpnI digestion and resistant to SpeI, AseI, HinfI and TfiI digestion (Figure S8B). These results indicated that Nham_0569 was only partially expressed in its native host. The modification sequences of MTases are summarized to Table 1. The MoDMP hosts and difficult-to-transform bacteria showed similar DNA methylation patterns based on the REase digestion analysis, but the DNA from MoDMP hosts have more modification sites than corresponding difficult-to-transform bacte- ria. And this was mainly caused by the limited expression of some MTases in their native hosts, especially some prophage-derived MTases, i.e., BAMTA208_6525, BCE_0393 and Nham_0569, which are multi-specific MTases. When co-expressed, BCE_0393, BCE_0365, BCE_4605, BCE_5606 and BCE_5607 protected all of the pMK4 plasmid from AvaII and BceAI digestion, protected most of the pMK4 plasmids from Fnu4HI, TseI, BbvI, HaeIII, EaeI, HpaII and NlaIV cleavage, provided pMK4 partial protection from HhaI digestion and provided pHCMC05 full protection from BssHII cleavage (Figure S7B). However, pMK4 plasmid prepared from the B. cereus ATCC 10987 strain was only resistant to TseI, BbvI, AvaII and BceAI digestion, and partially resistant to Fnu4HI digestion (Figure S7B), indicating that BCE_0393 is not com- pletely expressed in its native host. Discussion MM without 5-FU was used as a control medium. doi:10.1371/journal.pgen.1002987.g006 Figure 6. Inactivation of upp in B. amyloliquefaciens TA208 using an integration plasmid that underwent the MoDMP pipeline. (A) Schematic representation of the recombination event. The MoDMP prepared pWYE748 plasmid will recombine with the chromosome without being degraded by the REases. (B) PCR analysis of the B. amyloliquefaciens TA208 strain and the upp mutant BS043 strain using primers WB605 and WB606. (C) Verification of the loss of uracil phosphoribosyltransferase activity in B. amyloliquefaciens BS043. Strain BS043 could grow on MM plates containing 10 mM 5-FU while the TA208 strain could not. MM without 5-FU was used as a control medium. doi:10.1371/journal.pgen.1002987.g006 reported to restrict DNA containing Dam and Dcm methylation; for example, B. anthracis could be transformed by DNA from an E. coli dam dcm mutant strain but not by DNA from E. coli host strains with the wild type alleles [37,38]. Additionally, DNA prepared from the E. coli SCS110 strain was more accessible to Corynebac- terium glutamicum than DNA from E. coli hosts with Dam and Dcm [39]. However, not all difficult-to-transform bacteria behave like this. Bacteria lacking functional Type IV REases could be transformed by DNA prepared from E. coli hosts with Dam, Dcm or EcoKI, albeit at a low efficiency. Currently, it has been shown that the B. cereus ATCC 10987 strain does not restrict DNA with Dam and Dcm methylation. It has also been reported that the plasmids methylated in E. coli TOP10 cells using the MTases of the target bacteria can allow for the genetic manipulation of Bifidobacterium breve [40]. Ryan et al. showed that the bbe02 and bbq67 loci limited the transformation of Borrelia burgdorferi by shuttle vector DNA prepared from E. coli, irrespective of its Dam, Dcm or EcoKI methylation status [41]. The N. hamburgensis X14 strain used in this study may restrict DNA with Dcm methylation; the Staphylococcus aureus to efficiently accept foreign DNA [36]. In this study, a strategy has been developed to mimic the DNA methylation patterns of the difficult-to-transform bacteria in a modified E. coli strain. To achieve this goal, active MTases from the difficult-to-transform bacteria were co-expressed in an E. coli host lacking all of the characterized R-M systems and orphan MTases. reported to restrict DNA containing Dam and Dcm methylation; for example, B. anthracis could be transformed by DNA from an E. Discussion Genetic transformation of bacteria harboring multiple R-M systems has been problematic using conventional methods. It has been long recognized that the exogenous MTases over-expressed in E. coli could modify DNA in vivo and protect them from digestion by their cognate REases [31]. Strategies based on this fact have been developed to overcome the restriction barrier of bacteria, including in vitro or in vivo plasmid modification prior to transformation [32,33], heat inactivation of the REases [34] or gene knock-outs [35]. However, it has been reported that the inactivation of the SauI Type I R-M system is insufficient for For N. hamburgensis X14, Nham_0569 could protect DNA from cleavage by at least 10 REases sensitive to m6A modification, i.e., DpnI (GAmTC), DpnII (GATC), PvuII (CAGCTG), SspI (AATATT), SpeI (ACTAGT), MfeI (CAATTG), NlaIII (CATG), AseI (ATTAAT), HinfI (GANTC) and TfiI (GAWTC), but full protection was not achieved (Figure S8A). Therefore, Nham_0569 might be a multi-specific enzyme harboring at least eight modification sites, i.e., GATC, CAGCTG, AATATT, ACTAGT, CAATTG, CATG, ATTAAT and GANTC, or a new member of September 2012 \| Volume 8 \| Issue 9 \| e1002987 September 2012 \| Volume 8 \| Issue 9 \| e1002987 PLOS Genetics \| www.plosgenetics.org 8 A Pipeline for Overcoming Restriction Barrier Figure 6. Inactivation of upp in B. amyloliquefaciens TA208 using an integration plasmid that underwent the MoDMP pipeline. (A) Schematic representation of the recombination event. The MoDMP prepared pWYE748 plasmid will recombine with the chromosome without being degraded by the REases. (B) PCR analysis of the B. amyloliquefaciens TA208 strain and the upp mutant BS043 strain using primers WB605 and WB606. (C) Verification of the loss of uracil phosphoribosyltransferase activity in B. amyloliquefaciens BS043. Strain BS043 could grow on MM plates containing 10 mM 5-FU while the TA208 strain could not. MM without 5-FU was used as a control medium. doi:10.1371/journal.pgen.1002987.g006 Figure 6. Inactivation of upp in B. amyloliquefaciens TA208 using an integration plasmid that underwent the MoDMP pipeline. (A) Schematic representation of the recombination event. The MoDMP prepared pWYE748 plasmid will recombine with the chromosome without being degraded by the REases. (B) PCR analysis of the B. amyloliquefaciens TA208 strain and the upp mutant BS043 strain using primers WB605 and WB606. (C) Verification of the loss of uracil phosphoribosyltransferase activity in B. amyloliquefaciens BS043. Strain BS043 could grow on MM plates containing 10 mM 5-FU while the TA208 strain could not. Discussion hamburgensis X14 strain was used as a negative control. doi:10.1371/journal.pgen.1002987.g007 Figure 7. Expression of GFP in N. hamburgensis X14. The N. hamburgensis X14 strain was transformed with pWYE561 prepared from a MoDMP host. (A) Green fluorescence was observed in transformed N. hamburgensis X14 (lower row), but not in untransformed bacteria (upper row). (B) Determination of the proportion of transformed bacteria by flow cytometry analysis (lower). Bacterial culture of the N. hamburgensis X14 strain was used as a negative control. doi:10.1371/journal.pgen.1002987.g007 plasmid-borne putative Type IV R-M system Nham_4502- Nham_4503 has been annotated in the REBASE database [5], though its activity and specificity remain unclear. Nham_3845 (NhaXI) has been reported to be a fused enzyme harboring both restriction and modification subunits, but the m6A or m4C modification activity was not detected [44]; in this study the MTase activity was not detected either. It might be that Nham_3845 modified DNA in ways other than methylation, which could not be detected using immunoblot assays. Nham_3845 (NhaXI) has been reported to be a fused enzyme harboring both restriction and modification subunits, but the m6A or m4C modification activity was not detected [44]; in this study the MTase activity was not detected either. It might be that Nham_3845 modified DNA in ways other than methylation, which could not be detected using immunoblot assays. As shown in the upper right panel in Figure S9, expression of exogenous MTases in E. coli would result in methylation of chromosomal DNA, and Mrr, McrA and McrBC would recognize and cleave the DNA with foreign patterns, making the strain inviable or resulting in poor MTase expression [42,43]. Therefore, an E. coli strain lacking all of the known R-M systems and orphan MTases was generated with MTases expressed. The plasmids prepared from this host could escape the REases that recognize unmethylated DNA or DNA methylated in foreign patterns (lower right panel in Figure S9). The MoDMP concept could greatly improve genetic transformation efficiency. The contribution of individual MTases to genetic transformation was not evaluated in this study because a shuttle plasmid containing all of the MTase recognition sequences cannot be defined. A MTase that does not modify one particular plasmid might be useful when other plasmids are to be used. Therefore, to make a universal system for all plasmids, all of the identified active MTases were employed for MoDMP. Discussion coli dam dcm mutant strain but not by DNA from E. coli host strains with the wild type alleles [37,38]. Additionally, DNA prepared from the E. coli SCS110 strain was more accessible to Corynebac- terium glutamicum than DNA from E. coli hosts with Dam and Dcm [39]. However, not all difficult-to-transform bacteria behave like this. Bacteria lacking functional Type IV REases could be transformed by DNA prepared from E. coli hosts with Dam, Dcm or EcoKI, albeit at a low efficiency. Currently, it has been shown that the B. cereus ATCC 10987 strain does not restrict DNA with Dam and Dcm methylation. It has also been reported that the plasmids methylated in E. coli TOP10 cells using the MTases of the target bacteria can allow for the genetic manipulation of Bifidobacterium breve [40]. Ryan et al. showed that the bbe02 and bbq67 loci limited the transformation of Borrelia burgdorferi by shuttle vector DNA prepared from E. coli, irrespective of its Dam, Dcm or EcoKI methylation status [41]. The N. hamburgensis X14 strain used in this study may restrict DNA with Dcm methylation; the The protocol for genetic transformation of difficult-to-transform bacteria using a plasmid prepared in a different E. coli host is diagramed in Figure S9. As indicated in strain B. amyloliquefaciens TA208, DNA from the E. coli hosts with Dam and Dcm contains methylated bases in GAmTC and CCmWGG sequences but is not methylated at the recognition sequences of the host Type I–III REases; this DNA would then be recognized by Type I–IV REases in the target bacteria (upper left panel in Figure S9). Plasmids prepared from dam dcm EcoKI mutant E. coli would make the strains transformable at a low efficiency due to the plasmids being able to avoid restriction by the Type IV REases in the target bacterium (lower left panel in Figure S9). Many bacterial species have been September 2012 \| Volume 8 \| Issue 9 \| e1002987 PLOS Genetics \| www.plosgenetics.org 9 A Pipeline for Overcoming Restriction Barrier Figure 7. Expression of GFP in N. hamburgensis X14. The N. hamburgensis X14 strain was transformed with pWYE561 prepared from a MoDMP host. (A) Green fluorescence was observed in transformed N. hamburgensis X14 (lower row), but not in untransformed bacteria (upper row). (B) Determination of the proportion of transformed bacteria by flow cytometry analysis (lower). Bacterial culture of the N. September 2012 \| Volume 8 \| Issue 9 \| e1002987 Discussion The orphan MTase BAMTA208_06715, which lacks a counterpart REase, was also used in the MoDMP pipeline of B. amyloliquefaciens TA208. Orphan MTases, such as CcrM, may participate in methylation-directed DNA mismatch repair [45]. Methylated DNA could potentially escape inspection from the host mismatch repair machinery and eventually exhibit an elevated transformation performance, hence the use of BAMTA208_06715 in the MoDMP pipeline. Recently, four REases from the B. cereus ATCC 10987 strain have been cloned and characterized, namely BceSI, BceSII, BceSIII and BceSIV [15]. Only faint and non-specific hybridiza- tion blots were observed using the antibodies against m6A and m5C for the MTase of BceSI (BCE_1018) in this study; these faint blots may be caused by the vagaries of dot blot approaches. It might also be that BCE_1018 modifies the DNA in a way other than methylation, such as hydroxymethylation or glucosyl- hydroxymethylation. BCE_1018 was not included in the down- stream MoDMP application. Nevertheless, the highest transfor- mation efficiency was achieved using the plasmids modified by six other MTases and was within the acceptable range for gene knock- out experiments (107 CFU/mg DNA). This efficiency may be caused by the low abundance and the weak REase activity of BceSI in strain B. cereus ATCC 10987, as described by Hegna et al. [23]. BceSI was induced only when the strain B. cereus ATCC 10987 was grown in the presence of exogenous DNA. The use of DNA mimic protein Ocr (overcome classical restriction) alongside the plasmid (TypeOne Restriction Inhibitor, Epicentre; [46]) which specifically inhibits Type I REase activity, also enhances transformation efficiency in bacterial species [47]. Combination of this method with the MoDMP pipeline could further elevate transformation performance in strains which are difficult to transform. Recently, a novel R-M system has been shown to phosphorothioate DNA, preventing the degradation of the DNA by its REase counterparts [48]. The MoDMP concept may also be adapted to those bacteria restricting unphosphor- othioated DNA. September 2012 \| Volume 8 \| Issue 9 \| e1002987 PLOS Genetics \| www.plosgenetics.org 10 A Pipeline for Overcoming Restriction Barrier In conclusion, we devised a system in E. coli that mimics the DNA methylation patterns of bacterial strains harboring multiple R-M systems. Eventually, the R-M barrier of three represented bacterial strains were overcome, including Gram positive, Gram negative, chemoheterotrophs and chemoautotrophs. The adapt- ability of this pipeline to different bacterial groups suggests a universal potential. Southwestern dot blot assay for methylation activity The DNA methylation activity of the putative MTases was analyzed using a southwestern dot blot assay as described previously [18]. Total genomic DNA from the E. coli EC135 strains expressing individual or multiple MTases was prepared using a DNeasy Blood and Tissue Kit (Qiagen). DNA concentra- tions were determined using a Nanodrop 2000C spectrophotom- eter (Thermo Scientific). The DNA was then denatured at 100uC for 3 min and immediately cold shocked in an ice-water bath. Samples were spotted onto Protran BA85 nitrocellulose membrane (Whatman) and fixed by UV cross-linking. The membrane was blocked in 5% non-fat milk and incubated with rabbit antisera against DNA containing m6A at a dilution of 1:10,000 (New England Biolabs), rabbit antisera against m4C at a dilution of 1:10,000 (New England Biolabs), or a mouse monoclonal antibody against m5C diluted 1:20,000 (Zymo Research). After washing, the membrane was incubated with secondary goat anti-rabbit or anti- mouse antibodies conjugated with horseradish peroxidase (HRP) (Jackson ImmunoResearch) at a dilution of 1:10,000. The blots were visualized using the ECL prime Western blotting detection Materials and Methods The characterization of m6dA was performed on an Agilent 6520 Accurate-Mass QTOF LC/MS system (Agilent Technologies) equipped with an electrospray ionization (ESI) source. 30 mL of the samples were injected to the Agilent 1200 HPLC using an Agilent Zorbax Extend-C18 1.8 mm 2.1650 mm column with the column temperature kept at 35uC. Water with 0.1% formic acid and methanol were used as mobile phases A and B, respectively, with a flow rate of 0.2 mL/min. The following gradient was used: 0% B for 3.0 min, increase to 60% B in 4.5 min, 60–95% B over 2.5 min, 95% B for 5 min, and then decreased to 0% B over 0.5 min prior to re- stabilization of 14.5 min before the next injection. LC–MS assays for m6dA To obtain the nucleoside samples of genomic DNA for LC-MS analysis, 30 mg of DNA prepared from the E. coli EC135 strain or strains expressing MTases were digested to deoxynucleosides with 50 U of DNA Degradase Plus (Zymo Research); the digestion was carried out in 100 mL volume at 37uC for 18 h. The m6dA standard was purchased from Santa Cruz Biotechnology. Discussion This protocol is very fast; a MoDMP plasmid can be generated in less than one week using the S. cerevisiae assembler, if the MTase activity assay step is omitted and the putative MTases are cloned and expressed directly. We expect that the pipeline will be applicable to other strains of known genome sequence that are resistant to genetic transformation. reagent (GE Healthcare), and DNA methylation signals were exposed to Kodak X-Ray film. For quantification of the hybridization signals, the films were scanned and the gray scale of the spots was quantified using Quantity One (Bio-Rad). After normalization, the values were plotted as bar charts. Cloning and expression of putative MTases Cloning and expression of putative MTases Putative MTase encoding genes were retrieved from the REBASE database [5]. Genes were PCR amplified and ligated into pBAD43. Individual genes that encode the methylation and specificity subunits of BCE_0839–BCE_0842 system were joined to operons using Splicing by Overlapping Extension (SOE) PCR. All recombinant plasmids were verified by sequencing before use. The E. coli EC135 strain was transformed with pBAD43 plasmids encoding MTase genes. Single colonies were used to inoculate LB medium and cultured until an OD600 reading of 0.2 was reached, and then arabinose was added to a final concentration of 0.2% to induce MTase expression. Expression was induced overnight at 30uC. One-step assembly of multiple active MTase genes One step assembly of multiple active MTase genes Multiple MTase genes were rapidly assembled by taking advantage of the high DNA recombination activity in S. cerevisiae [49]. The CEN6 replicon was added to pBAD43 followed by TRP1 allele from pDDB78 at the ClaI site to yield pWYE724; the addition of these elements enables replication and screening in S. cerevisiae. The active MTase genes were amplified using PCR primers that contained 50 bp of overlapping sequence to the adjacent gene from their corresponding pBAD43 plasmids. S. cerevisiae DAY414 was transformed with the DNA fragments encoding the active MTases from the individual bacterial strains and the pWYE724 plasmid linearized at the EcoRI and SalI loci. S. cerevisiae DAY414 was then selected for tryptophan autotrophy on synthetic complete (SC) medium lacking tryptophan. S. cerevisiae transformation was performed using the lithium acetate method [50]. Plasmids were rescued into E. coli TOP10 cells as described by Robzyk et al [51]. All recombinant plasmids were verified by restriction digestion and DNA sequencing before subsequent use. The plasmids carrying multiple MTase genes from B. amylolique- faciens TA208, B. cereus ATCC 10987 and N. hamburgensis X14 were named pM.Bam, pM.Bce and pM.Nham, respectively. Strains and plasmids The strains E. coli TOP10 and EC135 were used for the cloning and expression of the MTases. S. cerevisiae DAY414 was used for in vivo assembly of the MTase genes. The plasmid pBAD43 was used for the cloning and expression of individual MTases, and pWYE724 was used for co-expression of multiple MTases. Several E. coli-Bacillus shuttle plasmids were used for MoDMP procedure evaluation purposes in the B. amyloliquefaciens TA208 and B. cereus ATCC 10987 strains. Inactivation of upp in B. amyloliquefaciens TA208 was performed with pWYE748. Expression of the GFP variant gfpmut3a in N. hamburgensis was carried out using pBBR1- MCS5. The strains and plasmids used in this study are listed in Table S1. The MS data were collected in positive ionization mode with nitrogen supplied as the nebulizing and drying gas. The temperature of the drying gas was set at 300uC. The flow rate of the drying gas and the pressure of the nebulizer were 600 L/h and 25 psi, respectively. The fragmentor and capillary voltages were kept at 90 and 3,500 V, respectively. Full-scan spectra were acquired over a scan range of m/z 80–1000 at 1.03 spectra/s. Determination of the modification sequences of MTases Determination of the modification sequences of MTases The modified plasmid DNA was challenged by the cognate REases to determine the modification sequences of the cloned MTases. To facilitate the identification, the high-copy plasmid pMK4 was transformed to E. coli EC135 harboring individual or multiple MTase genes, and in vivo methylated pMK4 plasmids were prepared and challenged by the cognate REases after linearization by REases that have sole cutting sites in pMK4 (NcoI, EcoRI, SpeI or BamHI). The pMK4 plasmids prepared from E. coli EC135, and the plasmids from B. amyloliquefaciens TA208 or B. cereus ATCC 10987 was used as the negative and positive controls in the experiments of individual MTase and multiple MTases, respectively. The plasmids pWYE690 and pHCMC02 were tested for their resistance to BclI cleavage conferred by BAMTA208_6525 when it was expressed individu- ally and co-expressed due to the lack of BclI site in pMK4. For the same reason, pWYE699 and pHCMC05 were used in testing the protection conferred by BCE_0393 from BssHII cleavage. Electroporation of B. amyloliquefaciens TA208 was performed using the combined cell-wall weakening and cell-membrane fluidity disturbing procedure described previously [17]. Since the broad-host-range plasmid derivative pWYE561 showed a low copy number in E. coli, pMK4 was also used in identification of the modification sites of the MTases from strain N. hamburgensis X14. Genomic DNA of the strain was used as a control for co-expressed MTases. y g p p y [ ] The N. hamburgensis X14 strain was grown in DSMZ 756a medium (1.5 g/L yeast extract, 1.5 g/L peptone (BD Biosciences), 2 g/L NaNO2, 0.55 g/L sodium pyruvate, 1 mL/L trace element solution (33.8 mg/L MnSO4?H2O, 49.4 mg/L H3BO3, 43.1 mg/ L ZnSO4?7H2O, 37.1 mg/L (NH4)6Mo7O24, 97.3 mg/L FeS- O4?7H2O and 25 mg/L CuSO4?5H2O) and 100 mL/L stock solution (0.07 g/L CaCO3, 5 g/L NaCl, 0.5 g/L MgSO4?7H2O, 1.5 g/L KH2PO4), pH 7.4) at 28uC in the dark until reaching an OD600 of 0.1. The cells were then harvested by centrifugation at 8000 g at 4uC for 10 min and washed four times with ice-cold 10% glycerol. The cells were resuspended in 10% glycerol at a 1,000-fold greater concentration compared to that of the original culture volume. The cell suspension (90 mL) was mixed with 150 ng of the pWYE561 plasmid and electroporated with an ECM399 electroporator at 1.2 kV. In vivo methylation of plasmids The pWYE748 plasmid was transformed into the E. coli EC135 strain harboring pM.Bam. After induction of MTase expression, 1 mg of the pWYE748 plasmid was transferred to B. amyloliquefa- ciens TA208, and the cells were selected for chloramphenicol resistance. Positive clones were verified by PCR and sequencing using primers (WB605 and WB606) specific to the flanking sequences of the homologous arms. The upp knock-out strain B. amyloliquefaciens BS043 was validated by growth on MM plates supplemented with 10 mM 5-FU [26] and 100 mg/L adenosine. All of the PCR primers used in this study are listed in Table S2. Various shuttle and integrative plasmids were transformed into the E. coli EC135 strains carrying MTase encoding genes. MTase expression was then induced by incubation with 0.2% arabinose at 30uC to allow the in vivo methylation of these plasmids. Determination of the modification sequences of MTases The cells were washed into 100 mL 756a medium and recovered at 28uC with gentle shaking for one day. The bacteria were then grown in the presence of 20 mg/mL gentamycin for one day. The bacterial culture was used at a ratio of 1:100 to inoculate fresh 756a medium containing antibiotics and was shaken at 180 rpm at 28uC. After about three weeks, the culture became turbid. The bacterial culture was subcultured once more to enrich for transformed cells and took one week to reach an OD600 of 0.1. The culture was tested for contamination microscopically and by streaking the culture onto LB plates. Successful transformation of strain X14 was verified by plasmid preparation using the Plasmid Mini Kit (OMEGA Bio- tek), PCR amplification of gfpmut3a and plasmid rescue. Expression A Pipeline for Overcoming Restriction Barrier of GFP was observed using a Leica TCS SP2 confocal laser scanning microscope (Leica Microsystems), and the ratio of fluorescent cells was determined using a BD FACS Calibur flow cytometer (BD Biosciences). A 216 bp promoter region of the Nham_3450 gene was PCR amplified from the genome of the N. hamburgensis X14 strain and joined to gfpmut3a by SOE-PCR. The resulting GFP expression cassette was ligated into pBBR1-MCS5 at the SalI and PstI sites to yield pWYE561. Transformation of difficult-to-transform bacteria Transformation of B. cereus ATCC 10987 was carried out as described previously with the following modifications [24]. The B. cereus ATCC 10987 strain was cultured in LB medium until the culture reached an OD600 of 0.2 and was then incubated on ice for 10 min. Cells were harvested by centrifugation at 8,000 g at 4uC for 10 min. After washing four times with ice-cold transformation buffer (10% sucrose, 15% glycerol, 1 mM Tris-HCl, pH 8.0), the electro-competent cells were resuspended in 1/125 volume of the original culture. The cells (90 mL) were mixed with 100 ng of the column-purified plasmids and loaded into a pre-chilled 1 mm gap cuvette. After a brief incubation on ice, the cells were shocked with a 2.1 kV pulse generated by a BTX ECM399 electroporator (Harvard Apparatus). The cells were immediately diluted with 1 mL NCMLB medium (17.4 g/L K2HPO4, 11.6 g/L NaCl, 5 g/ L glucose, 10 g/L tryptone (Oxoid), 5 g/L yeast extract (Oxoid), 0.3 g/L trisodium citrate, 0.05 g/L MgSO4?7H2O, 69.2 g/L mannitol and 91.1 g/L sorbitol, pH 7.2) and incubated at 37uC for 3 h to allow the expression of the antibiotic resistance genes. Aliquots of the recovery mix were spread onto LB plates supplemented with 5 mg/mL chloramphenicol or 10 mg/mL kanamycin and cultured overnight at 37uC. Construction of integration plasmids and GFP– expressing plasmids The homologous DNA sequences flanking the upp gene of B. amyloliquefaciens TA208 (641 bp upstream and 669 bp downstream) and the chloramphenicol resistance gene of pMK4 were amplified and joined using SOE-PCR. This cassette was ligated into the pMD19-T vector (Takara) and verified by DNA sequencing. The resulting plasmid was named pWYE748. September 2012 \| Volume 8 \| Issue 9 \| e1002987 PLOS Genetics \| www.plosgenetics.org 11 A Pipeline for Overcoming Restriction Barrier A Pipeline for Overcoming Restriction Barrier Relative intensities shown are averages of three replicates 6 SD. (TIF) normalized to that of 150 ng of E. coli TOP10 DNA (100%); for m4C, signals of the spots were normalized to that of 150 ng of E. coli EC135 DNA expressing BAMTA208_06525 (100%); for m5C, signals of the spots were normalized to that of 150 ng of E. coli EC135 DNA expressing BAMTA208_06715 (100%). (B) Hybrid- ization intensity of the DNA in vivo methylated by the MTases from N. hamburgensis X14. For m6A, signals of the spots were normalized to that of 150 ng of E. coli TOP10 DNA (100%); for m4C, signals of the spots were normalized to that of 150 ng of M.BamHI in vivo methylated E. coli EC135 DNA (100%); for m5C, signals of the spots were normalized to that of 150 ng of M.AluI in vivo methylated E. coli EC135 DNA (100%). Relative intensities shown are averages of three replicates 6 SD. (TIF) Figure S3 Growth curve of E. coli EC135 strains expressing Nham_0569, Nham_0803 and Nham_3225. The E. coli EC135 strains harboring pWYE712 (Nham_0569), pWYE714 (Nham_0803) and pWYE719 (Nham_3225) were cultured until an OD600 reading of 0.2 was reached, and then arabinose was added to a final concentration of 0.2% to induce MTase expression. The E. coli EC135 strain harboring the empty vector pBAD43 was used as the control. (TIF) Figure S4 Assembly of MTase genes. (A) MoDMP plasmids visualized on agarose gels. (B) EcoRV digestion of pM.Bam. (C) HindIII digestion of pM.Bce. (D) BamHI digestion of pM.Nham. The digestion patterns of the pM.Bam, pM.Bce and pM.Nham plasmids correspond with the theoretical patterns (B–D). (TIF) Figure S8 The modification sequences analysis of MTases from N. hamburgensis X14 expressed individually and co-expressed. (A) Analysis of the modification sequences of MTases expressed individually. The pMK4 plasmids prepared from the E. coli EC135 strains expressing individual MTases (identified in the text above the gel images) was challenged by cognate REases (identified in the text above the gel images). In each two-lane-grouped REase case, unmethylated pMK4 plasmids from E. coli EC135 were used as the control in the left lane, and methylated pMK4 plasmids prepared from the EC135 strains expressing individual MTases were used in the right lane. Prior to REase challenging, the pMK4 plasmids were linearized by REases (identified beneath the gel images) with sole recognition sites in it. A Pipeline for Overcoming Restriction Barrier (B) Confirmation of the modification sequences of MTases when co-expressed. The pMK4 plasmids prepared from E. coli EC135 (left lane in each REase case) or the strain EC135 harboring pM.Nham (right lane in each REase case) were challenged by cognate REases. The genomic DNA of N. hamburgensis X14 was used as the controls. M, DNA marker; U, undigested. The proportion of plasmids resistant to REase digestion is marked by asterisks. (TIF) Figure S5 Rescue of pWYE561 from transformed N. hamburgen- sis X14. (A) Original pWYE561 plasmid from E. coli TOP10 and rescued pWYE561 from N. hamburgensis X14. (B) Original and rescued pWYE561 digested by SalI and PstI. The length of pBBR1MCS5 backbone and promoter-gfpmut3a is noted. (TIF) Figure S6 The modification sequences analysis of MTases from strain B. amyloliquefaciens TA208 expressed individually and co- expressed. (A) Analysis of the modification sequences of MTases when expressed individually. The pMK4 plasmids prepared from E. coli EC135 strains expressing individual MTases (identified in the text above the gel images) was challenged by cognate REases (identified in the text above the gel images). In each two-lane- grouped REase case, unmethylated pMK4 plasmids from E. coli EC135 were used as the control in the left lane, and methylated pMK4 plasmids prepared from the EC135 strains expressing individual MTases were used in the right lane. Prior to REase challenging, the pMK4 plasmids were linearized by REases (identified beneath the gel images) with sole recognition sites. In the test of BclI site modified by BAMTA208_06525, EcoRI- linearized pWYE690 plasmid was challenged by BclI, and mock- treated linearized pWYE690 was used as the control. (B) Confirmation of the modification sequences of MTases when co- expressed. pMK4 plasmids prepared from the E. coli EC135 strain harboring pM.Bam (left lane in each REase case) or from the B. amyloliquefaciens TA208 strain (right lane in each REase case) were challenged by cognate REases. In the BclI case, pHCMC02 from E. coli EC135 and the strain harboring pM.Bam were challenged, with the genomic DNA of the B. amyloliquefaciens TA208 strain used as the control. M, DNA marker; U, undigested. The proportion of plasmids resistant to REase digestion is marked by asterisks; the proportion of plasmids partially digested by REases is marked by arrowheads. Figure S9 Model of how the MoDMP protocol results in the transformation of difficult-to-transform bacteria. Plasmid DNA prepared from an E. Supporting Information Figure S1 Generation and verification of the E. coli strain EC135. (A) PCR analysis of dcm deletion in the E. coli EC067 strain using primers WB064 and WB065; E. coli TOP10 strain was used as a control. (B) Digestion of chromosomal DNA with BstNI and PspGI for verification of dcm inactivation in the E. coli EC067, EC132 and EC135 strains; E. coli TOP10 strain was used as a control. (C) Survival ratio of E. coli EC067 and EC132 (recA+) strains when challenged with nalidixic acid (NA). (D) PCR analysis of dam deletion in the E. coli EC135 strain using primers WB062 and WB063; The E. coli EC132 strain was used as a control. (E) DpnI and DpnII digestion of chromosomal DNA for verification of dam inactivation in the E. coli EC135 strain; E. coli TOP10, EC067 and EC132 strains were used as controls. (F) Sensitivity of the E. coli EC135 strain to 2-aminopurine (2-AP) exposure; E. coli TOP10, EC067 and EC132 strains were used as controls. (TIF) Figure S2 Plots of hybridization intensity in dot blots. The spots of dot blot assay for individual MTases (Figure 1) were quantified and plotted. (A) Hybridization intensity of the DNA in vivo methylated by the MTases from the B. amyloliquefaciens TA208 and B. cereus ATCC 10987 strains. For m6A, signals of the spots were PLOS Genetics \| www.plosgenetics.org September 2012 \| Volume 8 \| Issue 9 \| e1002987 12 A Pipeline for Overcoming Restriction Barrier Figure S7 The modification sequences analysis of MTases from strain B. cereus ATCC 10987 expressed individually and co- expressed. (A) Analysis of the modification sequences of MTases expressed individually. The pMK4 plasmids prepared from the E. coli EC135 strains expressing individual MTases (identified in the text above the gel images) was challenged by cognate REases (identified in the text above the gel images). In each two-lane- grouped REase case, unmethylated pMK4 plasmids from E. coli EC135 were used as the control in the left lane, and methylated pMK4 plasmids prepared from the EC135 strains expressing individual MTases were used in the right lane. Prior to REase challenging, the pMK4 plasmids were linearized by REases (identified beneath the gel images) with sole recognition sites in it. In the test of BssHII site modified by BCE_0393, SpeI-linearized pWYE699 plasmid was challenged by BssHII, and mock-treated linearized pWYE699 was used as the control. (B) Confirmation of the modification sequences of MTases when co-expressed. The pMK4 plasmids prepared from the E. coli EC135 strain harboring pM.Bce (left lane in each REase case) or from B. cereus ATCC 10987 (right lane in each REase case) were challenged by cognate REases. In the BclI case, pHCMC05 from E. coli EC135 and the strain harboring pM.Bce were challenged, and the genomic DNA of B. cereus ATCC 10987 was used as the control. M, DNA marker; U, undigested. The proportion of plasmids resistant to REase digestion is marked by asterisks; the proportion of plasmids partially digested by REases is marked by arrowheads. (TIF) normalized to that of 150 ng of E. coli TOP10 DNA (100%); for m4C, signals of the spots were normalized to that of 150 ng of E. coli EC135 DNA expressing BAMTA208_06525 (100%); for m5C, signals of the spots were normalized to that of 150 ng of E. coli EC135 DNA expressing BAMTA208_06715 (100%). (B) Hybrid- ization intensity of the DNA in vivo methylated by the MTases from N. hamburgensis X14. For m6A, signals of the spots were normalized to that of 150 ng of E. coli TOP10 DNA (100%); for m4C, signals of the spots were normalized to that of 150 ng of M.BamHI in vivo methylated E. coli EC135 DNA (100%); for m5C, signals of the spots were normalized to that of 150 ng of M.AluI in vivo methylated E. coli EC135 DNA (100%). References (2011) Characterization of Type II and III restriction-modification systems from Bacillus cereus strains ATCC 10987 and ATCC 14579. J Bacteriol 194: 49–60. 39. Okibe N, Suzuki N, Inui M, Yukawa H (2011) Efficient markerless gene replacement in Corynebacterium glutamicum using a new temperature-sensitive plasmid. J Microbiol Methods 85: 155–163. 16. Zhang G, Deng A, Xu Q, Liang Y, Chen N, et al. (2011) Complete genome sequence of Bacillus amyloliquefaciens TA208, a strain for industrial production of guanosine and ribavirin. J Bacteriol 193: 3142–3143. 40. O’Connell Motherway M, O’Driscoll J, Fitzgerald GF, Van Sinderen D (2009) Overcoming the restriction barrier to plasmid transformation and targeted mutagenesis in Bifidobacterium breve UCC2003. Microbial Biotechnology 2: 321–332. 17. Zhang G, Bao P, Zhang Y, Deng A, Chen N, et al. (2011) Enhancing electro- transformation competency of recalcitrant Bacillus amyloliquefaciens by combining cell-wall weakening and cell-membrane fluidity disturbing. Anal Biochem 409: 130–137. 41. Rego ROM, Bestor A, Rosa PA (2011) Defining the plasmid-borne restriction- modification systems of the lyme disease spirochete Borrelia burgdorferi. J Bacteriol 193: 1161–1171. 18. Kong H, Lin LF, Porter N, Stickel S, Byrd D, et al. (2000) Functional analysis of putative restriction–modification system genes in the Helicobacter pylori J99 genome. Nucleic Acids Res 28: 3216–3223. 42. Raleigh EA, Murray NE, Revel H, Blumenthal RM, Westaway D, et al. (1988) McrA and McrB restriction phenotypes of some E. coli strains and implications for gene cloning. Nucleic Acids Res 16: 1563–1575. g 19. Zhou H, Wang Y, Yu Y, Bai T, Chen L, et al. (2012) A non-restricting and non- methylating Escherichia coli strain for DNA cloning and high-throughput conjugation to Streptomyces coelicolor. Curr Microbiol 64: 185–190. 43. Waite-Rees PA, Keating CJ, Moran LS, Slatko BE, Hornstra LJ, et al. (1991) Characterization and expression of the Escherichia coli Mrr restriction system. J Bacteriol 173: 5207–5219. 20. Marinus MG (2010) DNA methylation and mutator genes in Escherichia coli K- 12. Mutat Res 705: 71–76. 44. Morgan RD, Dwinell EA, Bhatia TK, Lang EM, Luyten YA (2009) The MmeI family: type II restriction–modification enzymes that employ single-strand modification for host protection. Nucleic Acids Res 37: 5208–5221. 21. Le T, Kim K-P, Fan G, Faull KF (2011) A sensitive mass spectrometry method for simultaneous quantification of DNA methylation and hydroxymethylation levels in biological samples. Anal Biochem 412: 203–209. 45. References Gene 10: 219–22 p g gy 9. Arp DJ, Bottomley PJ (2006) Nitrifiers: more than 100 years from isolation to genome sequences. Microbe 1: 229–234. 32. Donahue JP, Israel DA, Peek RM, Blaser MJ, Miller GG (2000) Overcoming the restriction barrier to plasmid transformation of Helicobacter pylori. Mol Microbiol 37: 1066–1074. 10. Starkenburg SR, Larimer FW, Stein LY, Klotz MG, Chain PSG, et al. (2008) Complete genome sequence of Nitrobacter hamburgensis X14 and comparative genomic analysis of species within the genus Nitrobacter. Appl Environ Microbiol 74: 2852–2863. 33. Groot MN, Nieboer F, Abee T (2008) Enhanced transformation efficiency of recalcitrant Bacillus cereus and Bacillus weihenstephanensis isolates upon in vitro methylation of plasmid DNA. Appl Environ Microbiol 74: 7817–7820. 34. Van der Rest ME, Lange C, Molenaar D (1999) A heat shock following electroporation induces highly efficient transformation of Corynebacterium glutamicum with xenogeneic plasmid DNA. Appl Microbiol Biotechnol 52: 541– 545. 11. Rasko DA, Ravel J, Økstad OA, Helgason E, Cer RZ, et al. (2004) The genome sequence of Bacillus cereus ATCC 10987 reveals metabolic adaptations and a large plasmid related to Bacillus anthracis pXO1. Nucleic Acids Res 32: 977–988. 12. Arnaud M, Chastanet A, De´barbouille´ M (2004) New vector for efficient allelic replacement in naturally nontransformable, low-GC-content, Gram-positive bacteria. Appl Environ Microbiol 70: 6887–6891. 35. Dong H, Zhang Y, Dai Z, Li Y (2010) Engineering Clostridium strain to accept unmethylated DNA. PLoS ONE 5: e9038. doi:10.1371/journal.pone.0009038 pp 13. Lindba¨ck T, Kolstø A-B (1997) A Bacillus cereus member of the SNF2 family. Microbiology 143: 171–174. 36. Veiga H, Pinho MG (2009) Inactivation of the SauI Type I restriction- modification system is not sufficient to generate Staphylococcus aureus strains capable of efficiently accepting foreign DNA. Appl Environ Microbiol 75: 3034–3038. 14. Lindba¨ck T, Økstad OA, Rishovd A-L, Kolstø A-B (1999) Insertional inactivation of hblC encoding the L2 component of Bacillus cereus ATCC 14579 haemolysin BL strongly reduces enterotoxigenic activity, but not the haemolytic activity against human erythrocytes. Microbiology 145: 3139–3146. y p g g pp 37. Marrero R, Welkos SL (1995) The transformation frequency of plasmids into Bacillus anthracis is affected by adenine methylation. Gene 152: 75–78. 38. Sitaraman R, Leppla SH (2012) Methylation-dependent DNA restriction in Bacillus anthracis. Gene 494: 44–50. 15. Xu S-y, Nugent RL, Kasamkattil J, Fomenkov A, Gupta Y, et al. Table S2 Primers used for PCR. (DOC) Table S2 Primers used for PCR. (DOC) A Pipeline for Overcoming Restriction Barrier coli host with Dam, Dcm and EcoKI are methylated at GAmTC, CCmWGG and EcoKI recognition sequences, but not at the recognition sequence of the REases from the difficult-to-transform bacteria. This DNA would be degraded by Type I–IV REases upon transformation into the bacteria (upper-left quarter). Expression of exogenous MTases from the difficult-to-transform bacteria strains in the E. coli host with McrA, McrBC and Mrr would cause degradation of the host’s chromosomal DNA (upper-right quarter). Plasmids pre- pared from dam- dcm- EcoKI- E. coli hosts can be transformed at low efficiencies because the restriction imposed by Type IV REases has been relieved (lower-left quarter). When the plasmids were prepared from the E. coli strain lacking known R-M systems and (TIF) PLOS Genetics \| www.plosgenetics.org September 2012 \| Volume 8 \| Issue 9 \| e1002987 13 A Pipeline for Overcoming Restriction Barrier A Pipeline for Overcoming Restriction Barrier orphan MTases while expressing MTases of the bacteria, the plasmids showed similar modification patterns to the bacteria and can be transformed at high efficiencies (lower-right quarter). (TIF) Author Contributions Text S1 Supporting Information Methods and Supporting Information Results. The construction and validation of E. coli EC135 is described in detail. Conceived and designed the experiments: GZ YC TW. Performed the experiments: GZ WW AD ZS. Analyzed the data: GZ WW AD ZS YZ YL YC TW. Wrote the paper: GZ YC TW. Acknowledgments We are grateful to Ciara´n Condon at CNRS UPR9073 for valuable suggestions. We thank Richard Morgan at New England Biolabs for the kind gifts of antisera against m6A and m4C, John Beckwith for pBAD43, Mingchun Li for S. cerevisiae DAY414 and pDDB78, and BGSC for various shuttle plasmids. We thank Chunli Li for the help of confocal laser scanning microscopy, Tong Zhao for flow cytometry, and Jianzhong Li at Agilent Technologies China for the help of LC–MS analysis. Table S1 Strains and plasmids used in this study. (DOC) References 1. Bottone EJ (2010) Bacillus cereus, a volatile human pathogen. Clin Microbiol Rev 23: 382–398. 25. 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https://openalex.org/W3089062014	https://da.lib.kobe-u.ac.jp/da/kernel/90007547/90007547.pdf	English	null	Traditional craftspeople are not copycats: Potter idiosyncrasies in vessel morphogenesis	PloS one	2,020	cc-by	10,353	Traditional craftspeople are not copycats: Potter idiosyncrasies in vessel morphogenesis Gandon, Enora Nonaka, Tetsushi Endler, John A. Coyle, Thelma Bootsma, Reinoud J. Gandon, Enora Nonaka, Tetsushi Endler, John A. Coyle, Thelma Bootsma, Reinoud J Kobe University Repository : Kernel PDF issue: 2024-10-24 Traditional craftspeople are not copycats: Potter idiosyncrasies in vessel morphogenesis (Citation) PLoS ONE,15(9):e0239362-e0239362 (Issue Date) 2020-09-22 Gandon, Enora Nonaka, Tetsushi Endler, John A. Coyle, Thelma Bootsma, Reinoud J. Kobe University Repository : Kernel PDF issue: 2024-10-24 PDF issue: 2024-10-24 Traditional craftspeople are not copycats: Potter idiosyncrasies in vessel morphogenesis (Citation) ( (Citation) PLoS ONE,15(9):e0239362-e0239362 (Citation) PLoS ONE,15(9):e0239362-e0239362 (Issue Date) 2020-09-22 (Resource Type) journal article (Version) Version of Record PLOS ONE RESEARCH ARTICLE Enora GandonID1☯, Tetsushi Nonaka2☯, John A. EndlerID3, Thelma Coyle4, Reinoud J. BootsmaID4* Enora GandonID1☯, Tetsushi Nonaka2☯, John A. EndlerID3, Thelma Coyle4, Reinoud J. BootsmaID4* 1 Institute of Archaeology, University College London, London, United Kingdom, 2 Graduate School of Human Development and Environment, Kobe University, Kobe, Japan, 3 Centre for Integrative Ecology, School of Life & Environmental Sciences, Deakin University, Waurn Ponds, Australia, 4 Institut des Sciences du Mouvement, Aix Marseille Universite´, CNRS, Marseille, France a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 ☯These authors contributed equally to this work. * reinoud.bootsma@univ-amu.fr Editor: Mark Nielsen, University of Queensland, AUSTRALIA Editor: Mark Nielsen, University of Queensland, AUSTRALIA Received: April 21, 2020 Accepted: September 6, 2020 Published: September 22, 2020 Copyright: © 2020 Gandon 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 Ceramics are quintessential indicators of human culture and its evolution across genera- tions of social learners. Cultural transmission and evolution theory frequently emphasizes apprentices’ need for accurate imitation (high-fidelity copying) of their mentors’ actions. However, the ensuing prediction of standardized fashioning patterns within communities of practice has not been directly addressed in handicraft traditions such as pottery throwing. To fill this gap, we analysed variation in vessel morphogenesis amongst and within tradi- tional potters from culturally different workshops producing for the same market. We demon- strate that, for each vessel type studied, individual potters reliably followed distinctive routes through morphological space towards a much-less-variable common final shape. Our results indicate that mastering the pottery handicraft does not result from accurately repro- ducing a particular model behaviour specific to the community’s cultural tradition. We pro- vide evidence that, at the level of the elementary clay-deforming gestures, individual learning rather than simple imitation is required for the acquisition of a complex motor skill such as throwing pottery. OPEN ACCESS Citation: Gandon E, Nonaka T, Endler JA, Coyle T, Bootsma RJ (2020) Traditional craftspeople are not copycats: Potter idiosyncrasies in vessel morphogenesis. PLoS ONE 15(9): e0239362. https://doi.org/10.1371/journal.pone.0239362 Editor: Mark Nielsen, University of Queensland, AUSTRALIA (Rights) © 2020 Gandon 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. (URL) https://hdl.handle.net/20.500.14094/90007547 PLOS ONE Introduction During the forming phase thinning the clay walls brings out a preliminary roughout as the vessel rises from its base, while the final form is attained after the ultimate shaping operations. In fashion- ing the vessel, potters successively deploy several distinctive hand positions for contact with the clay [10]. Addressing the influence of cultural setting on wheel-throwing practices, in ear- lier work we analysed hand position sequences as observed in French, Indian and Nepalese potting communities [13–16]. Here we analysed the throwing process by tracking the potter-induced morphological changes in the clay body, from its initial pre-formed stage following centring and opening operations, up to the moment that the final form is reached. We emphasize that vessel mor- phogenesis, as studied here, allows capturing the essential result of each manual fashioning gesture in terms of the change in clay shape brought about by contact between the hands and clay. In so doing, it provides an integrative view of the potter’s shaping actions. Our analyses focused on variation in morphological development of the clay body, both amongst different potters and within individual potters, as they each repeatedly threw customary vessel types. As a long-standing behavioural tradition, wheel-throwing clearly entails social learning and transmission [1, 17, 18]. Typical for such behavioural traditions, the seven Indian potters par- ticipating in the present study had learned the craft as apprentices guided by elder mentors who had previously learned it themselves in much the same way within the same tradition. If, like their elders, these potters had learned how to throw a particular type of vessel through high-fidelity copying, that is, by accurately imitating each step in their mentors’ way of doing [4–6, 19–21], within each community of practice [22] fashioning patterns would be culturally standardized. In the framework of the present study, such high-fidelity copying would there- fore be expected to give rise to low variation among potters from the same community in the throwing process and, thereby, in the resulting final vessels forms. Ethnoarchaeological studies examining the degree of product standardization (quantified by metric variability) in same-type vessel assemblages have pinpointed the essential role of eco- nomic specialization, with intensive production allowing potters to fully develop their skill. Highly standardized vessels can indeed only be consistently delivered by expert specialists, that is, by highly-skilled potters [23–25]. Introduction Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Social learning allows cultural traditions to persist over generations [1]. Among the processes underlying cultural transmission [2, 3], action imitation has been suggested as being particu- larly important and, perhaps, specific to humans [4, 5]. Indeed, accurate imitation of their accomplished elders’ way of doing, commonly denoted high-fidelity copying, is argued to allow learners to “ratchet up” existing knowledge, thereby reducing or eliminating the need to rediscover an effective way of solving each problem [6]. While presented as a general principle, to our knowledge the operation of high-fidelity copying has so far not been experimentally explored in behavioural traditions involving complex motor skills such as pottery and other handicrafts. Here we bring a motor behaviour perspective to bear on the question of whether potters, having learned the skill from elders within their community, could have done so by Funding: This project has received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska- Curie grant agreement No 793451 awarded to E.G.. 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 there are no competing interests. PLOS ONE \| https://doi.org/10.1371/journal.pone.0239362 September 22, 2020 1 / 18 September 22, 2020 PLOS ONE Traditional craftspeople are not copycats imitation. We do this by focussing on the observable results of potters’ clay-deforming techni- cal gestures, Pottery, the craft of making container objects out of clay, is an ancient human tradition [7]. Gradually superseding earlier coiling, the introduction of the throwing technique exploiting the fast-rotating wheel, dating back at least to the Middle Bronze II era (1750 BC), allowed sub- stantial gains in production rate and product regularity [8, 9]. At the same time, however, it made the fashioning process considerably more difficult to master and thereby longer to learn [10, 11]. Starting from the moment the lump of clay is placed on the rotating wheel, the throwing process itself is characterized by an invariant sequence of general operations [9, 10, 12]. During the pre-forming phase the potter first centres the mass of clay on the wheel and subsequently sets the stage for the forming process by opening (hollowing) the centred lump of clay. PLOS ONE \| https://doi.org/10.1371/journal.pone.0239362 September 22, 2020 Introduction Becoming an expert in any area requires at least ten years of extended, deliberate practice [26] and genuinely mastering the craft of wheel-throwing is no exception to this rule [10]. The road to expertise is therefore long and demanding. Rather than relying on invariably rehearsing the same gestures, deliberate practice has been argued to give rise to exploration and reorganizations of the skill [27, 28], so as to find suitable individualized solutions to the problems posed by the task at hand. While being capable of consistently obtaining the same desired result, experts thus typically reveal individual differ- ences (‘styles’) between them in their way of doing [29–33]. For the present purposes, this inter-individual functional motor equivalence perspective [28, 34–36] suggests that the low PLOS ONE \| https://doi.org/10.1371/journal.pone.0239362 September 22, 2020 2 / 18 September 22, 2020 PLOS ONE Traditional craftspeople are not copycats variability that is expected in the final forms thrown by expert potters need not be associated with similarly low between-potter variability in the throwing process: different experts are likely to have found different routes to the same end result. In the context of the present study, hypotheses about between-potter and within-potter var- iation in final vessel forms are therefore fairly clear-cut. We explore two alternative hypotheses: (1) if each potter learns by high-fidelity copying of a model, then throughout the forming pro- cess the degree of among-potter variation should be relatively small and of the same order as within-potter variation; (2) if each potter learns by deliberate practicing, then the degree of among-potter variation should be greater than within-potter variation and this pattern should be strongest at the earlier stages of morphogenesis, with among-potter variations decreasing as the vessel nears its final form. In order to test these hypotheses we requested potters to produce series of customary traditional forms, expected to yield final assemblages with low overall shape variability for each vessel type. Earlier experimental work [37, 38] indicated that for experts these final pottery assemblages would be characterized by low within-potter variability, resulting in subtle but discernible individual potter signatures. The field experiment allowing to address these issues took place in the northern India region of Uttar Pradesh. In this region the pottery handicraft is a traditional activity, with the skill being transmitted vertically within endogamous castes that produce standardized tradi- tional objects in mass production [10, 39]. Results In order to capture the morphological development towards the final form, for each vessel thrown we digitized its outline from video frames after every clay-deforming manual fashion- ing gesture. Fig 1 shows clay form as a function of time for two out of the five Money-bank tri- als performed by each of the seven potters (see S2 Fig for the full data set). Introduction Video-based data acquisition took place in the workshops of two potting communities working and living in the same village and mainly dif- ferentiated by the use of different wheels. The Prajapati potters used a hand-operated, high- inertia stick-wheel, while the Multani Kumhar potters used a foot-operated, low-inertia kick- wheel. Both communities used the same soft grey clay to produce a similar repertoire of tradi- tional vessel types for a common market. All seven participating potters were confirmed experts with more than ten years of experi- ence on the task. Among the four potters from the Prajapati workshop (referred to as AR, BA, GA and KA), GA and KA were father and son; GA and AR were uncle and nephew. There was no close family relationship between the three potters from the Multani Kumhar workshop (KD, NA and YA). For the present purposes, each community was asked to select three pre- ferred traditional pottery types, to be thrown by each potter in five specimens with self-selected quantities of clay. Prajapati potters selected the Money-bank, Handiya, and Kullar, while Mul- tani Kumhar potters selected the Money-Bank, Handi, and Kulfi (see S1 Fig for graphical rep- resentations of these traditional vessel types). Therefore, in the following analyses we concentrated most of our attention on the Money-banks thrown by all participating potters. Corroborative evidence from the analyses of the four other vessels types is mainly presented as supporting information. PLOS ONE \| https://doi.org/10.1371/journal.pone.0239362 September 22, 2020 Final vessel dimensions and size development As allowed by our protocol based on self-selected quantities of clay, final vessel size varied over potters, with Multani potters YA and NA producing, respectively, the largest and the smallest Money-banks. ANOVAs on absolute final vessel dimensions (see Table 1) corroborated the observation that final Money-bank size was not community-specific, as all F’s (1,5) < 1 and 3 / 18 PLOS ONE \| https://doi.org/10.1371/journal.pone.0239362 September 22, 2020 PLOS ONE Traditional craftspeople are not copycats Fig 1. Morphological development of Money-bank vessels as a function of time. Two examples (trials 3 and 4) for Money-banks thrown by each of the Prajapati potters (top panel: GA, KA, BA, AR) and Multani potters (bottom panel: KD, NA, YA). Successive outlines on the timelines represent the vessel form after each fashioning gesture of the potter, from the initial pre-formed shape (t = 0) up to the final vessel shape. Size scale (height) is indicated on the y-axis. Note the slightly different time scale for BA. https://doi.org/10.1371/journal.pone.0239362.g001 Fig 1. Morphological development of Money-bank vessels as a function of time. Two examples (trials 3 and 4) for Money-banks thrown by each of the Prajapati potters (top panel: GA, KA, BA, AR) and Multani potters (bottom panel: KD, NA, YA). Successive outlines on the timelines represent the vessel form after each fashioning gesture of the potter from the initial pre formed shape (t 0) up to the final essel shape Size scale (height) is indicated on the a is Fig 1. Morphological development of Money-bank vessels as a function of time. Two examples (trials 3 and 4) for Money-banks thrown by each of the Prajapati potters (top panel: GA, KA, BA, AR) and Multani potters (bottom panel: KD, NA, YA). Successive outlines on the timelines represent the vessel form after each fashioning gesture of the potter, from the initial pre-formed shape (t = 0) up to the final vessel shape. Size scale (height) is indicated on the y-axis. Note the slightly different time scale for BA. https://doi.org/10.1371/journal.pone.0239362.g001 PLOS ONE \| https://doi.org/10.1371/journal.pone.0239362 September 22, 2020 4 / 18 PLOS ONE Traditional craftspeople are not copycats Table 1. Absolute dimensions of final vessels. Means and coefficients of variation (100SD/M, between parentheses) of height (H), maximal diameter (MD) and exterior surface area (ESA) of the final forms across trials for the traditional vessels thrown by each potter. Final vessel dimensions and size development Vessel type Prajapati Multani Kumhar GA KA BA AR KD NA YA Money-bank H (cm) 16.3 (5.4) 18.4 (2.3) 14.5 (5.9) 15.2 (2.0) 15.9 (2.7) 12.9 (4.8) 17.7 (3.1) MD (cm) 14.3 (4.2) 13.9 (2.4) 12.3 (8.7) 12.8 (4.5) 14.2 (3.4) 11.1 (1.3) 17.4 (2.1) ESA (cm2) 543.3 (8.3) 596.9 (4.6) 422.8 (13.2) 466.9 (8.5) 500.6 (5.5) 330.9 (6.2) 745.8 (4.1) H (cm) 13.9 (7.0) 13.8 (2.0) 12.7 (2.7) 13.2 (2.5) Handiya MD (cm) 15.3 (8.3) 16.6 (1.1) 14.9 (3.9) 14.8 (1.9) ESA (cm2) 587.0 (14.7) 640.0 (3.2) 525.4 (7.4) 557.0 (5.1) H (cm) 14.7 (5.2) 12.5 (3.5) 14.4 (3.4) 14.2 (2.7) Kullar MD (cm) 12.0 (3.6) 11.4 (2.0) 12.7 (3.5) 12.1 (5.2) ESA (cm2) 513.8 (7.8) 413.3 (5.9) 528.8 (7.8) 473.1 (8.1) H (cm) 12.1 (3.7) 12.7 (2.3) 14.3 (4.0) Handi MD (cm) 17.4 (1.5) 17.8 (1.2) 20.5 (1.9) ESA (cm2) 580.0 (3.6) 640.5 (2.7) 842.5 (5.5) H (cm) 9.2 (3.4) 8.33 (4.1) 9.7 (3.6) Kulfi MD (cm) 12.5 (2.3) 12.3 (3.6) 14.3 (2.0) ESA (cm2) 346.3 (3.5) 320.6 (6.7) 445.1 (4.4) Table 1. Absolute dimensions of final vessels. Means and coefficients of variation (100SD/M, between parentheses) of height (H), maximal diameter (MD) and exterior surface area (ESA) of the final forms across trials for the traditional vessels thrown by each potter. effect sizes were small (η2 g = 0.024, 0.056 and 0.005, respectively, for height, maximal diameter and exterior surface area). Fig 2. Growth curves of Money-bank exterior surface area. Symbols and curves indicate exterior surface area (ESA) for individual potters (Prajapati GA, KA, BA and AR; Multani Kumhar KD, NA and YA), represented as a function of 20-bin normalized time, from the pre-formed initial form (time bin 1) up to the final vessel form (time bin 20). Curves represent modeled vessel growth curves. Each symbol represents the mean ESA of the trials with one or more form points falling within a particular time bin. Error bars represent ±1 SE. https://doi.org/10.1371/journal.pone.0239362.g002 Fig 2. Growth curves of Money-bank exterior surface area. Symbols and curves indicate exterior surface area (ESA) for individual potters (Prajapati GA, KA, BA and AR; Multani Kumhar KD, NA and YA), represented as a function of 20-bin normalized time, from the pre-formed initial form (time bin 1) up to the final vessel form (time bin 20). Curves represent modeled vessel growth curves. https://doi.org/10.1371/journal.pone.0239362.t001 effect sizes were small (η2 g = 0.024, 0.056 and 0.005, respectively, for height, maximal diameter and exterior surface area). Final vessel dimensions and size development Each symbol represents the mean ESA of the trials with one or more form points falling within a particular time bin. Error bars represent ±1 SE. Fig 2. Growth curves of Money-bank exterior surface area. Symbols and curves indicate exterior surface area (ESA) for individual potters (Prajapati GA, KA, BA and AR; Multani Kumhar KD, NA and YA), represented as a function of 20-bin normalized time, from the pre-formed initial form (time bin 1) up to the final vessel form (time bin 20). Curves represent modeled vessel growth curves. Each symbol represents the mean ESA of the trials with one or more form points falling within a particular time bin. Error bars represent ±1 SE. https://doi.org/10.1371/journal.pone.0239362.g002 5 / 18 PLOS ONE \| https://doi.org/10.1371/journal.pone.0239362 September 22, 2020 PLOS ONE Traditional craftspeople are not copycats Considerable differences between potters were also observed for the time taken to fashion a vessel of a given type and for the number of manual fashioning gestures deployed (visible in Fig 1 as the number of digitized outlines on the trial timelines). Throwing larger Money-banks did not, however, systematically require more time, as indicated by the non-significant Pear- son correlation between throwing duration and final vessel exterior surface area (r33 = 0.07, P = 0.68, 95% CI = [-0.269, 0.394]). Throwing duration was in fact shortest for Prajapati potter AR and Multani potter KD (M ± SD respectively 61.2 ± 2.8 s and 62.9 ± 6.0 s) and longest for Prajapati potter BA (155.8 ± 37.9 s). Throwing larger Money-banks was positively related to the number of fashioning gestures deployed (r33 = 0.41, P = 0.012, 95% CI = [0.092, 0.656]), varying from 5 or 6 for Prajapati potter AR to 10 or 11 for Multani potter YA and 8 to 12 for Prajapati potter BA. As anticipated, within-potter variation in final vessel size was smaller than between-potter variation; for example, for Money-bank exterior surface area F(6,28) = 64.21, P < 0.0001. This observation, corroborated by the small (on average 4.2%) within-potter coefficients of varia- tion of the absolute vessel dimensions (Table 1), supports the general finding that the outcome of expert behaviour is highly reproducible over repeated trials [15, 25, 40]. Final vessel dimensions and size development As can be seen from Fig 1, the Money-bank size development over time was generally char- acterized by a rapid initial increase, followed by a small decrease that gradually levelled out, sometimes reversing to a slight increase, towards the end of the throwing process. This pattern is clearly evident in the development of exterior surface area in the (20-bin) time-normalized representation of Fig 2 and could thus effectively be captured by an orthogonal 3rd-order poly- nomial growth curve model. Although inclusion of a random effect of individual-potter inter- cept significantly improved the model fit (likelihood ratio test: χ2(1) = 469.75, P < 0.0001), between-potter growth-curve differences could not be fully reduced to differences in initial size. Time trends in size space were potter specific, as demonstrated by a further improvement of the model (likelihood ratio test: χ2(9) = 144.21, P = 0.0001) by inclusion of a random effect for the slope of the three time-terms. We found no evidence for a community fixed effect (like- lihood ratio test: χ2(4) = 7.91, P = 0.0949). PLOS ONE \| https://doi.org/10.1371/journal.pone.0239362 September 22, 2020 Final vessel shape Notwithstanding these differences in size, all potters produced clearly identifiable (i.e., type- specific) traditional forms (e.g., see last image on each row in Fig 1 for Money-banks). To quantify pure shape, we performed elliptical Fourier analysis on all digitized clay outlines and normalized the resulting Fourier coefficients to the first harmonic to correct for size [13, 37]. The full set of size-corrected Fourier coefficients was then subjected to a Principal Component (PC) analysis. For all evolving vessels over 80% of the total shape variance was captured by the first three PCs. Hence, the development of shape could adequately be traced out in this shared 3D shape-space. The five final Money-bank shapes thrown by each of the seven potters are presented in the left panels of Fig 3 (see S3 Fig for the other traditional vessel types). Despite the apparent over- all similarity in shape across vessels produced by different potters, permutation tests revealed statistically significant heterogeneity among individuals for each traditional type (Table 2, “Final Shape” columns). These results demonstrated that different potters produced vessels with more morphometric variation among than within potters, both over communities and within communities. Corroborating the results of earlier work [37, 38], we therefore conclude that expert potters imprint subtle but identifiable individual (shape) signatures even on typical traditional types of vessel produced for a common consumer market. 6 / 18 PLOS ONE \| https://doi.org/10.1371/journal.pone.0239362 September 22, 2020 September 22, 2020 PLOS ONE Traditional craftspeople are not copycats Fig 3. Development of Money-bank morphology in shape space. Right panels: Development of vessel morphology is represented as trajectories through 3D shape space, from the initial pre-formed shape (open circles) to the final shape (open squares), for the Money-bank vessels thrown by all seven potters (b), by Prajapati potters GA, KA, BA and AR (d) and by Multani Kumhar potters KD, NA and YA (f). Individual potters are colour-coded. For each potter mean initial shape is represented by an outline centered on the position indicated by an asterisk. Left panels: Zoom on final vessel shapes (open squares) revealing subtle between-potter differences. For each potter mean final shape is represented by an outline centered on the position indicated by an asterisk (a, c and e). Fig 3. Development of Money-bank morphology in shape space. Final vessel shape Right panels: Development of vessel morphology is represented as trajectories through 3D shape space, from the initial pre-formed shape (open circles) to the final shape (open squares), for the Money-bank vessels thrown by all seven potters (b), by Prajapati potters GA, KA, BA and AR (d) and by Multani Kumhar potters KD, NA and YA (f). Individual potters are colour-coded. For each potter mean initial shape is represented by an outline centered on the position indicated by an asterisk. Left panels: Zoom on final vessel shapes (open squares) revealing subtle between-potter differences. For each potter mean final shape is represented by an outline centered on the position indicated by an asterisk (a, c and e). Fig 3. Development of Money-bank morphology in shape space. Right panels: Development of vessel morphology is represented as trajectories through 3D shape space, from the initial pre-formed shape (open circles) to the final shape (open squares), for the Money-bank vessels thrown by all seven potters (b), by Prajapati potters GA, KA, BA and AR (d) and by Multani Kumhar potters KD, NA and YA (f). Individual potters are colour-coded. For each potter mean initial shape is represented by an outline centered on the position indicated by an asterisk. Left panels: Zoom on final vessel shapes (open squares) revealing subtle between-potter differences. For each potter mean final shape is represented by an outline centered on the position indicated by an asterisk (a, c and e). https://doi.org/10.1371/journal.pone.0239362.g003 PLOS ONE \| https://doi.org/10.1371/journal.pone.0239362 September 22, 2020 7 / 18 PLOS ONE Traditional craftspeople are not copycats Table 2. Results (R2 and P-values) of the permutation tests performed on the size-corrected coefficients resulting from elliptical Fourier analyses of the final and initial pre-formed clay shapes for each traditional vessel type thrown by the four Prajapati (PR) and the three Multani Kumhar (MK) potters. For each vessel type, within-potter effects are based on five trials, except for the Handiya where one trial was missing for PR potter AR. Table 2. Results (R2 and P-values) of the permutation tests performed on the size-corrected coefficients resulting from elliptical Fourier analyses of the final and initial pre-formed clay shapes for each traditional vessel type thrown by the four Prajapati (PR) and the three Multani Kumhar (MK) potters. For each vessel type, within-potter effects are based on five trials, except for the Handiya where one trial was missing for PR potter AR. Table 2. Vessel morphogenesis Fig 1 shows that, except for potter BA, the Money-bank shaping processes (i.e., the morpholog- ical development) were fairly consistent over trials within individual potters (also see S2 Fig for the other vessel types). In contrast, we found large differences amongst potters in morpho- logical development towards each final vessel form. In throwing the Money-banks, for exam- ple, after the second fashioning gesture Multani potter YA invariably produced a large shape with a rounded sphere-like bottom and a wide, rimmed aperture on the top. Then, the widest part of the vessel (i.e., where the diameter is maximal) moved progressively upward after each successive gesture, followed by the closing of the aperture on the top, resulting in a large, almost spherical final shape. In contrast, after the second fashioning gesture Prajapati potter GA produced almost cylindrical shapes with a rim around the aperture. The vessel’s profile subsequently became rounder with each successive gesture. Multani Potter NA produced less pronounced cylindrical shapes after a few gestures. Prajapati potter KA and Multani potter KD produced distinct though both barrel-like shapes after the first gestures. KA consistently pro- duced elongated vessels, while KD produced wider vessels, with sharper curves at the maximal diameter height. Prajapati potter AR narrowed the aperture immediately after the second ges- ture, thereby rapidly approaching the final form. Idiosyncrasies in morphological routes (towards the final shape) appeared not only at inter- mediary stages but were already present before the onset of the forming phase (t0 in Fig 1). As can be seen from the right panels in Fig 3 for the Money-banks, the starting positions of the trajectories (corresponding to the shape products of the pre-forming phase) varied markedly over potters; the same phenomenon was observed for the other traditional types (see S3 Fig). Permutation tests confirmed that, for each of the traditional types thrown except Kullar, among-potter variation in pre-formed clay shape was significantly larger than within-potter variation, both over communities and within communities (see Table 2, “Pre-formed Shape” columns), corroborating the idea that the expert potters have their own idiosyncratic ways of shaping the visibly standard, traditional vessels. Despite the significant differences among pot- ters for both final shapes and pre-formed shapes, the individual-trial Euclidean distances from Idiosyncrasies in morphological routes (towards the final shape) appeared not only at inter- mediary stages but were already present before the onset of the forming phase (t0 in Fig 1). Final vessel shape Results (R2 and P-values) of the permutation tests performed on the size-corrected coefficients resulting from elliptical Fourier analyses of the final and initial pre-formed clay shapes for each traditional vessel type thrown by the four Prajapati (PR) and the three Multani Kumhar (MK) potters. For each vessel type, within-potter effects are based on five trials, except for the Handiya where one trial was missing for PR potter AR. Type Component Final Shape Pre-formed Shape df R2 P R2 P Money-bank (PR & MK) Potter 6 0.55 <0.0001 0.76 <0.0001 Residuals 28 0.45 0.24 Money-bank (PR) Potter 3 0.41 <0.0001 0.65 <0.0001 Residuals 16 0.59 0.35 Money-bank (MK) Potter 2 0.53 <0.0001 0.65 <0.0001 Residuals 12 0.47 0.35 Handiya (PR) Potter 3 0.39 0.0007 0.58 0.0006 Residuals 15 0.61 0.42 Kullar (PR) Potter 3 0.40 0.0005 0.24 0.1076 Residuals 16 0.60 0.76 Handi (MK) Potter 2 0.38 0.001 0.61 <0.0001 Residuals 12 0.62 0.39 Kulfi (MK) Potter 2 0.43 <0.0001 0.57 <0.0001 Residuals 12 0.57 0.43 https://doi.org/10.1371/journal.pone.0239362.t002 PLOS ONE \| https://doi.org/10.1371/journal.pone.0239362 September 22, 2020 Discussion The shaping process as characterized by vessel morphogenesis data invalidates the high- fidelity copying hypothesis and is highly consistent with the deliberate practicing hypothe- sis. While we did not identify individual potter mentors (except for Prajapati GA’s son KA and nephew AR), we stress that within the community-of-practice perspective [22] a cul- tural model is taken to be shared by the community rather than being specific to each indi- vidual member. Yet, for each of the different vessel types studied participants followed clearly distinctive, individual routes through morphological space towards the much less variable final shape. Within specific communities, such variation amongst potters in vessel morphogenesis was observed whether close family relationships between participants existed (as in the Prajapati community) or not (as in the Multani Kumhar community). In addition, our results indicated that the number of fashioning gestures and the time required to fashion the vessels also significantly differed over participants. Thus, the seven participat- ing potters demonstrated idiosyncratic fashioning styles that were equivalent for producing the same pottery types. Overall, these results provide empirical evidence in support of motor behaviour theory’s functional equivalence principle in the domain of pottery handicraft. By focusing on the observable outcome of the elementary clay-deforming gestures our results reveal that cultural transmission of handicrafts does not entail near-perfect replication of elders’ way of doing although it does replicate the intended shape consistently and very well. We suggest that, at least at the level of elementary gestures [41–43], the proposition that fidel- ity-copying would underlie the emergence of skilled crafting behaviour is in fact fallacious. Learning a complex motor skill requires active exploration of the constraints and opportunities offered by the environment for achieving the task at hand. To truly appreciate craftmanship it is therefore essential to realize that the difficulty of the task implies that it requires such indi- vidual skill learning to recur across generations [43]. This is not, of course, to say that cultural transmission plays no role in handicrafts. As highly specialized motor behaviours, handicrafts could hardly be acquired on the basis of individual learning alone [1]. The learning environ- ment itself is already clearly socially structured, with the presence of typical tools and materials, finished vessels, active potters, etcetera. Social learning and transmission are thus no doubt key mechanisms operating to retain craft traits over generations. Vessel morphogenesis As can be seen from the right panels in Fig 3 for the Money-banks, the starting positions of the trajectories (corresponding to the shape products of the pre-forming phase) varied markedly over potters; the same phenomenon was observed for the other traditional types (see S3 Fig). Permutation tests confirmed that, for each of the traditional types thrown except Kullar, among-potter variation in pre-formed clay shape was significantly larger than within-potter variation, both over communities and within communities (see Table 2, “Pre-formed Shape” columns), corroborating the idea that the expert potters have their own idiosyncratic ways of shaping the visibly standard, traditional vessels. Despite the significant differences among pot- ters for both final shapes and pre-formed shapes, the individual-trial Euclidean distances from PLOS ONE \| https://doi.org/10.1371/journal.pone.0239362 September 22, 2020 8 / 18 PLOS ONE Traditional craftspeople are not copycats the group mean in the 3D shape-space computed at the initial and final stages indicated that potters started from considerably different locations in shape-space but all converged onto closely neighbouring shape-space locations at the final stage for Money-bank (see Fig 4) as well as for the other traditional vessel types (see S4 Fig). A linear mixed model ANOVA on the individual-trial Euclidean distances from the group mean in shape space with fixed-effects for the Stage factor and a random effect for the intercept for individual potter revealed significant effects of Stage for all vessel types (P’s < 0.001, see Table 3). These results provide unequivocal evidence that potters reliably followed distinctive individual routes through morphological space towards a much less variable final shape. Taken together, the present results demonstrated that, in traditional wheel-throwing, the variation among potters in vessel morphogenesis (i.e., in the clay’s morphological transforma- tion routes) is substantially greater than the subtle potter-specific variation remaining in the final shapes produced. This is true for all five traditional vessel types and both potter commu- nities examined. PLOS ONE \| https://doi.org/10.1371/journal.pone.0239362 September 22, 2020 https://doi.org/10.1371/journal.pone.0239362.g004 https://doi.org/10.1371/journal.pone.0239362.g004 https://doi.org/10.1371/journal.pone.0239362.g004 Students of sociocultural learning have pointed out that when newcomers (whether they be children or apprentices) learn a craft or other cultural motor skill, the interaction with elders mastering the task guides participation in the practice and scaffolds the learning process [3, 22, 45, 46]. In so doing, the cultural environment provides social pointers that orient and channel the learner’s attention, allowing perceptuomotor exploration to occur over an optimal area of the task space [47–50]. This social channelling helps the learner to perceive the relevant task space properties and to progressively exploit them functionally through practice. The func- tional (i.e., effective) aspects of motor skill thereby acquired correspond to the bodily actions that cause the intended effects on the environment. Importantly, these functional aspects gen- erally do not correspond to observable body configurations and kinematics but rather to the fine tunings of interaction kinetics. In the case of pottery throwing the functional aspects cor- respond to the tuning of the pressure forces exerted by the hands so as to plastically deform particular areas of the clay body; in the case of stone knapping [42] or flaking [43] they corre- spond to the tuning of the hammer’s kinetic energy transferred onto particular locations of the handheld platform so as to detach a flake. What matters here is to acknowledge that the func- tional aspects of motor skill mastered by experts are not observable, nor truly verbalizable; they correspond to know-how, non-discursive embodied knowledge [51] that cannot be trans- mitted or copied independent of its application in the world [52]. The functional aspects of the task need to be discovered through individual, hands-on exploration [30]. What may be reproduced during social learning are the formal (i.e., non-effective) aspects of the skill. Contrary to functional aspects, formal aspects and their sequencing are observable and can be verbalized, captured in action recipes and characterized by words or images. At the level of the handicraft’s elementary gestures, such formal aspects correspond to body segment positions and kinematics. Importantly, these aspects do not determine the result of the skill as the functional aspects do. In the case of wheel-throwing the formal aspects related to the ele- mentary gestures notably include the hand positions successively used in fashioning a vessel. These hands positions do not determine the manual pressure forces exerted and thereby the vessel shape [13]. Discussion What we suggest is that the conceptualisation of cultural transmission by way of fidelity-copying poorly grasps these intri- cate mechanisms of skill learning [44]. PLOS ONE \| https://doi.org/10.1371/journal.pone.0239362 September 22, 2020 9 / 18 September 22, 2020 PLOS ONE Traditional craftspeople are not copycats LOS ONE Traditional craftspeople are not copycats PLOS ONE \| https://doi.org/10.1371/journal.pone.0239362 September 22, 2020 10 / 18 PLOS ONE Traditional craftspeople are not copycats Fig 4. Euclidean distances (in shape space) from group mean Money-bank shapes. Boxplots represent individual-trial Euclidean distance from the group mean shape at the initial pre-formed stage and at the final stage for the Money-bank vessels thrown (a) by all seven potters and (b) by Prajapati potters GA, KA, BA and AR and Multani Kumhar potters KD, NA and YA separately. Note that at each stage the pertinent group mean shapes differ over the groups considered. Individual potters are colour coded. The solid and dotted lines in the box indicate the medians and means of the data, respectively. https://doi.org/10.1371/journal.pone.0239362.g004 Yet, as revealed by personal ethnographic observations in different potting communities, mentors typically encourage their apprentices to carefully watch the hands posi- tions used so that they can reproduce them. The existence of culturally specific hand positions Table 3. Results (F, df and P-values) for the factor Stage of the linear mixed model analysis of the Euclidean dis- tance from the grand mean in shape space, at the initial pre-formed stage and at the final stage, for each tradi- tional vessel type thrown by the four Prajapati (PR) and the three Multani Kumhar (MK) potters. For each vessel type, analysis is based on five trials, except for the Handiya where one trial was missing for PR potter AR. Table 3. Results (F, df and P-values) for the factor Stage of the linear mixed model analysis of the Euclidean dis- tance from the grand mean in shape space, at the initial pre-formed stage and at the final stage, for each tradi- tional vessel type thrown by the four Prajapati (PR) and the three Multani Kumhar (MK) potters. For each vessel type, analysis is based on five trials, except for the Handiya where one trial was missing for PR potter AR. Table 3. Results (F, df and P-values) for the factor Stage of the linear mixed model analysis of the Euclidean dis- tance from the grand mean in shape space, at the initial pre-formed stage and at the final stage, for each tradi- tional vessel type thrown by the four Prajapati (PR) and the three Multani Kumhar (MK) potters. For each vessel type, analysis is based on five trials, except for the Handiya where one trial was missing for PR potter AR. Type df F (Stage) P Money-bank (PR+MK) 62 1465.2 <0.0001 Money-bank (PR) 35 359.7 <0.0001 Moneybank (MK) 26 33.81 <0.0001 Handiya (PR) 33 161.5 <0.0001 Kullar (PR) 35 77.28 <0.0001 Handi (MK) 26 116.37 <0.0001 Kulfi (MK) 26 31.18 <0.0001 11 / 18 PLOS ONE \| https://doi.org/10.1371/journal.pone.0239362 September 22, 2020 11 / 18 PLOS ONE \| https://doi.org/10.1371/journal.pone.0239362 September 22, 2020 September 22, 2020 PLOS ONE Traditional craftspeople are not copycats in French, Nepalese and Indian professional potting groups [14] attests that potters do repro- duce the hands positions they have observed during their learning. https://doi.org/10.1371/journal.pone.0239362.g004 Still, the co-existence of such culturally specific hand positions with both idiosyncratic and cross-cultural hand posi- tions indicates that potters do not limit their repertoire to the cultural hand positions observed in elders. As a cautionary remark, we emphasize that formal copying should not be conflated with the true learning of the skill. Without active engagement in deliberate practice to explore the task space, strict formal copying will not lead to the emergence of skill. However, formal copying of observable aspects of a mentor’s behaviour may be expected to serve as a social pointer usefully channelling the learner’s activity. Formal copying can thus be understood as a method of facili- tating the apprenticeship but not as a direct route to mastering the task constraints. Even when learning is facilitated by formal copying of a model, the necessary active exploration of the functional aspects of the task space will give rise to individual differences in skill ultimately acquired. As acknowledged by Forte [53], performance on a cultural motor skill may thus be understood as “a growing interaction between the transmission of knowledge and the develop- ment of manual practice” (p. 1). We conclude that, notwithstanding its apparent explanatory power, fidelity-copying does not form the principal basis of cultural transmission of handicraft and other activities rooted in complex motor skills. The finding of highly similar artefacts should not be taken as evidence for a highly standardized transformation process, since fidelity-copying is not the fundamental mechanism by which cultural motor skills are transmitted; the present results instead fit much better with the alternative scenario of culturally specific skills that takes into account the com- plexity of skill learning, in which individuals are given opportunities for discovering the func- tional requirements of the task and building up their own ways of coping with them. Continuity in behavioural traditions is in fact possible because each new generation of experts learns to control the end-result of the task (i.e., what they want to produce). Expert potters can accurately reproduce different vessel shapes, whether they are acquainted with them or not [54]. https://doi.org/10.1371/journal.pone.0239362.g004 This ability to control the characteristics of the final vessel thrown (including not only form but also resistance to collapse [55]) allows for the perpetuation and long-term develop- ment of potting traditions under the influence of a myriad of socio-economical and psycholog- ical factors, including production habits, market demand and social conformism among craftsmen of a given community of practice. By highlighting the necessity of extensive individual deliberate practice in the acquisition of technical motor skills, the present study underscores that variations detected in the material record should not be considered as uniquely resulting from perception and/or dexterity- related error in copying a model [17]; material variation in artifacts also results from inter- individual differences in solutions for equivalent motor problems, discovered during appren- ticeship. Another implication of our findings is that the relation between assemblage standard- ization and homogeneity of underlying manufacturing patterns is far from univocal. One should bear in mind that vessels falling into homogeneous geometrical types are not produced by culturally standardized fashioning patterns but by behaviours containing both cultural and individual features. The pool of individual behavioural variants existing in a community of practice is may constitute a source of change in artefacts. Procedure Potters from each workshop were asked to select three preferred traditional pottery types, to be thrown by each potter in their usual conditions of practice in five specimens with self- selected quantities of clay. Prajapati potters selected the Money-bank, Handiya, and Kullar, while Multani Kumhar potters selected the Money-Bank, Handi, and Kulfi (see S1 Fig for graphical representations of the vessel types). The experimental sessions were video-recorded under standardized conditions using a Panasonic NV-GS320 camcorder. The camera was fixed on a tripod with lens orientation cen- tred on the vertical rotation axis of the wheel. The camera was positioned at a height of 30 cm above the level of the wheel at a horizontal distance of 4–6 m. The lower edge of the video scene was aligned with the centre of the wheel. The zoom was adapted to fully cover a 36-cm high by 42-cm wide calibration object (inverted T-shape) placed on the wheel at the start of each recording. Of the total of 105 vessels thrown (seven potters, each throwing five specimens of three dif- ferent vessel types), one (a Handiya vessel thrown by Prajapati potter AR) could not be ana- lysed due to problems with the video-recording. Ethics statement The study consisted in non-invasive behavioural observations of potters in their habitual workshops. Potters gave informed written consent prior to participation and were paid for their participation according to the local rates of the profession. These observations were made in the framework of E.G.’s Ph.D. project at Aix-Marseille University (France). According to operative French law (1988 Huriet-Serusclat law, amended in 2004) on the protection of per- sons in biomedical research, such a protocol did not require the approval of an ethics commit- tee. The study was carried out in accordance with the ethical standards of the Declaration of Helsinki, the University of Aix-Marseille (E.G.’s affiliation at the time) and the Indian Anthro- pological Association. Participants Following visits during which the project was presented, participants were recruited within two traditional pottery workshops (one Prajapati and one Multani Kumhar) located in the PLOS ONE \| https://doi.org/10.1371/journal.pone.0239362 September 22, 2020 12 / 18 PLOS ONE Traditional craftspeople are not copycats same village in the Bulandshar district of the northern India state of Uttar Pradesh. In both communities, the throwing of the pots is traditionally performed by men only. After being duly informed, individual potters voluntarily decided to take part in the study. They were financially compensated for their participation. The participants were right-handed men over 25 years of age (Mean ± SD, Prajapati: 41.3 ± 14.9 yrs and Multani Kumhar: 33.7 ± 4.5 yrs) and had a minimum of ten years of wheel-throwing experience (Prajapati: 24.3 ± 14.5 yrs; Multani Kumhar: 21.0 ± 6.1 yrs). Potters from both participating workshops regularly produced at least five different kinds of everyday objects for the same local market, often in different sizes. PLOS ONE \| https://doi.org/10.1371/journal.pone.0239362 September 22, 2020 Data analysis For each trial, the images of the clay body profile after each fashioning gesture were extracted from the video frames (image resolution: 720 x 576 pixels; video sampling frequency: 25 fps). The first image captured the profile immediately following the (centring and opening) pre- forming phase and the last image captured the final profile; the intervening images captured the intermediate profiles during form development. This succession of profiles captured the vessel’s morphogenesis. The overall duration of the forming process and the total number of fashioning gestures per trial were also analysed as global variables describing the potters’ performances. From the images we extracted the 2D coordinates of the right-half of the cross-sectional profiles by tracing them out on a Cintiq 21UX Wacom1 (Kazo, Japan) tablet with integrated PLOS ONE \| https://doi.org/10.1371/journal.pone.0239362 September 22, 2020 13 / 18 PLOS ONE Traditional craftspeople are not copycats screen. All further analyses were performed using Matlab1 (MathWorks, Natick MA, USA). The profile coordinates were converted from pixels to centimetres using a calibration factor obtained from the digitized dimensions of the calibration object. The profiles were re-sampled to generate an equal number of (256) points at regular height intervals along the vertical (Y) axis and the resulting coordinates were smoothed with a low-pass filter [13, 54]. Because wheel-thrown vessels are typically axisymmetric, profiles were subsequently converted to full pot outlines by multiplying the horizontal (X) coordinates by -1 to create the corresponding left edge. Vessel dimensions were quantified by height, maximum diameter and exterior surface area. The latter was used as an overall measure of size. To capture the development of size, the five trials of each potter throwing the Money-bank were time-normalized by dividing the duration between the first and the last fashioning ges- ture of each trial into 20 equally spaced time bins, and by representing the time of each stage of morphogenesis as ith bin within which the observed shape occurred. Growth curve models with third-order orthogonal polynomials using the maximum likelihood estimates [56] were fitted to the development of exterior surface area. In order to quantify vessel shape, each outline was subjected to an elliptical Fourier trans- formation [13, 37, 57]. The resulting series of pairs of coefficients were normalized with respect to the first coefficients to correct for size differences [57, 58]. For graphical representation of the shape the size-corrected Fourier coefficients were subjected to a Principal Component (PC) analysis. Data analysis Since over 80% of the full-dataset (857 outlines) total variance was captured by the first three components (57.5%, 13.0% and 10.3%, respectively), each particular shape could thus be represented as a point in a unique 3D PC space, allowing qualitative (visual inspection) and quantitative (numerical) comparisons of shape similarities and differences. PLOS ONE \| https://doi.org/10.1371/journal.pone.0239362 September 22, 2020 Project administration: Enora Gandon. Resources: Thelma Coyle. Resources: Thelma Coyle. Software: Tetsushi Nonaka. Supervision: Enora Gandon, Reinoud J. Bootsma. Supervision: Enora Gandon, Reinoud J. Bootsma. Visualization: Tetsushi Nonaka, Reinoud J. Bootsma. Visualization: Tetsushi Nonaka, Reinoud J. Bootsma. Writing – review & editing: Enora Gandon, Tetsushi Nonaka, John A. Endler, Thelma Coyle, Reinoud J. Bootsma. Statistical analysis For all statistical tests the (two-sided) alpha level used was 0.05. Differences between commu- nities (Prajapati and Multani Kumhar) in height, maximum diameter and external surface area of the Money-bank vessels thrown were examined using Community x Trial ANOVAs with repeated measures on the last factor. Effect size of the Community factor was determined using generalized eta-squared (η2 g) [59]. Relationships between the size of the vessel thrown (using the external surface area measure), on the one hand, and throwing duration and num- ber of fashioning gestures deployed, on the other hand, were assessed using Pearson correla- tions and the associated 95% confidence intervals. In the growth model analysis of vessel size, the effects of adding the individual-potter random effects for the intercept and the slope, and the fixed effect of community on the improvement in model fit were tested using the likelihood ratio test which treats the change in a deviance measure (-2 log likelihood) as a chi-square sta- tistic [60]. Statistical differences among shapes were examined using permutation tests [61] on the normalized Fourier Coefficients scores for each of the five shape classes separately using the adonis function in the R package vegan [62]. This analysis, performed at the initial pre- formed and final stages of shape development, tested for heterogeneity of shapes among the potters within shape types; if significant this test indicates the presence of individual influences on shape. Using the lme function in the R package nlme [63], the proximity of locations in the 3D shape-space (as measured by the individual-trial Euclidean distances from the mean loca- tion in shape space) were modelled using a linear mixed model with a fixed-effect for the Stage factor (initial pre-formed stage vs. final stage) and a random intercept effect for individual pot- ter. A variance function (varIdent of nlme package) that allows different variances per stratum for each individual potter was used to model heteroscedasticity. The analysis was conducted PLOS ONE \| https://doi.org/10.1371/journal.pone.0239362 September 22, 2020 14 / 18 September 22, 2020 PLOS ONE Traditional craftspeople are not copycats for each of the five vessel types separately. For the Money-bank, the individual-trial Euclidean distance from the 7-potter overall mean as well as that from the mean locations of the Prajapati and Multani Kumhar communities were analysed separately. for each of the five vessel types separately. Statistical analysis For the Money-bank, the individual-trial Euclidean distance from the 7-potter overall mean as well as that from the mean locations of the Prajapati and Multani Kumhar communities were analysed separately. Author Contributions Author Contributions Conceptualization: Enora Gandon, Reinoud J. Bootsma. Formal analysis: Enora Gandon, Tetsushi Nonaka, John A. Endler, Reinoud J. Bootsma. Funding acquisition: Enora Gandon. Investigation: Enora Gandon. Methodology: Enora Gandon, Tetsushi Nonaka, John A. Endler, Thelma Coyle, Reinoud J. Bootsma. g Methodology: Enora Gandon, Tetsushi Nonaka, John A. Endler, Thelma Coyle, Reinoud J. Bootsma. Methodology: Enora Gandon, Tetsushi Nonaka, John A. Endler, Thelma Coyle, Reinoud J. Bootsma. Project administration: Enora Gandon. Supporting information Supporting information S1 Fig. Customary traditional vessel types thrown. (PDF) S2 Fig. Morphological development of all vessels thrown by each individual potter. 7 pages., 1 potter per page. (PDF) S3 Fig. Development of vessel morphology in shape space for other traditional vessel types. (PDF) S4 Fig. Euclidean distances (in shape space) from group mean shapes for other traditional vessel types. (PDF) Author Contributions Conceptualization: Enora Gandon, Reinoud J. Bootsma. Formal analysis: Enora Gandon, Tetsushi Nonaka, John A. Endler, Reinoud J. Bootsma. Funding acquisition: Enora Gandon. Investigation: Enora Gandon. Methodology: Enora Gandon, Tetsushi Nonaka, John A. Endler, Thelma Coyle, Reinoud J. Bootsma. Project administration: Enora Gandon. Resources: Thelma Coyle. Software: Tetsushi Nonaka. Supervision: Enora Gandon, Reinoud J. Bootsma. Visualization: Tetsushi Nonaka, Reinoud J. Bootsma. Writing – review & editing: Enora Gandon, Tetsushi Nonaka, John A. Endler, Thelma Coyle, Reinoud J. Bootsma. References 1. Boyd R, Richerson PJ, Henrich J. The cultural niche: why social learning is essential for human adapta- tion. Proc Natl Acad Sci USA. National Academy of Sciences; 2011; 108: 10918–10925. https://doi.org/ 10.1073/pnas.1100290108 PMID: 21690340 2 Schiffer MB Skibo JM Theory and Experiment in the Study of Technological Change Curr Anthropol pp g S1 Fig. Customary traditional vessel types thrown. (PDF) S2 Fig. Morphological development of all vessels thrown by each individual potter. 7 pages., 1 potter per page. (PDF) S3 Fig. Development of vessel morphology in shape space for other traditional vessel types. (PDF) S4 Fig. Euclidean distances (in shape space) from group mean shapes for other traditional vessel types. (PDF) References 1. Boyd R, Richerson PJ, Henrich J. The cultural niche: why social learning is essential for human adapta- tion. Proc Natl Acad Sci USA. National Academy of Sciences; 2011; 108: 10918–10925. https://doi.org/ 10.1073/pnas.1100290108 PMID: 21690340 2. Schiffer MB, Skibo JM. Theory and Experiment in the Study of Technological Change. Curr Anthropol. 1987; 28: 595–622. https://doi.org/10.1086/203601 3. Crown PL. The archaeology of crafts learning: Becoming a potter in the Puebloan Southwest. Annu Rev Anthropol. 2014; 43: 71–88. https://doi.org/10.1146/annurev-anthro-102313-025910 4. Tennie C, Call J, Tomasello M. Ratcheting up the ratchet: on the evolution of cumulative culture. 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https://openalex.org/W2938916495	https://europepmc.org/articles/pmc6518245?pdf=render	English	null	Real-Time Continuous Surveillance of Temperature and Flow Events Presents a Novel Monitoring Approach for Hospital and Healthcare Water Distribution Systems	International journal of environmental research and public health/International journal of environmental research and public health	2,019	cc-by	5,409	Received: 18 March 2019; Accepted: 7 April 2019; Published: 13 April 2019 Abstract: Within hospitals and healthcare facilities opportunistic premise plumbing pathogens (OPPPs) are a major and preventable cause of healthcare-acquired infections. This study presents a novel approach for monitoring building water quality using real-time surveillance of parameters measured at thermostatic mixing valves (TMVs) across a hospital water distribution system. Temperature was measured continuously in real-time at the outlet of 220 TMVs located across a hospital over a three-year period and analysis of this temperature data was used to identify ﬂow events. This real-time temperature and ﬂow information was then compared with microbial water quality. Water samples were collected randomly from faucets over the three-year period. These were tested for total heterotrophic bacteria, Legionella spp. and L. pneumophila. A statistically signiﬁcant association with total heterotrophic bacteria concentrations and the number of ﬂow events seven days prior (rs[865] = −0.188, p < 0.01) and three days prior to sampling (rs[865] = −0.151, p < 0.01) was observed, with decreased heterotrophic bacteria linked to increased ﬂushing events. Only four samples were positive for Legionella and statistical associations could not be determined; however, the environmental conditions for these four samples were associated with higher heterotrophic counts. This study validated a simple and eﬀective remote monitoring approach to identifying changes in water quality and ﬂagging high risk situations in real-time. This provides a complementary surveillance strategy that overcomes the time delay associated with microbial culture results. Future research is needed to explore the use of this monitoring approach as an indicator for diﬀerent opportunistic pathogens. Keywords: Legionella; Pseudomonas; non-tuberculous mycobacteria; healthcare; opportunistic waterborne pathogens; water quality; water management; public health Real-Time Continuous Surveillance of Temper and Flow Events Presents a Novel Monitoring Approach for Hospital and Healthcare Water Distribution Systems Harriet Whiley 1,* , Jason Hinds 2, James Xi 2 and Richard Bentham 1 Harriet Whiley 1,* , Jason Hinds 2, James Xi 2 and Richard Bentham 1 1 College of Science and Engineering, Flinders University, GPO Box 2100, Adelaide, SA Richard.Bentham@ﬂinders.edu.au 2 Enware Australia Pty Ltd., 11 Endeavour Road, Caringbah, NSW 2229, Australia; Jason.Hinds@enware.com.au (J.H.); James.Xi@enware.com.au (J.X.) * Correspondence: Harriet.Whiley@ﬂinders.edu.au; Tel.: +61-8-7221-8580 Harriet Whiley 1,* , Jason Hinds 2, James Xi 2 and Richard Bentham 1 1 College of Science and Engineering, Flinders University, GPO Box 2100, Adelaide, SA 5001, Australia; Richard.Bentham@ﬂinders.edu.au 2 Enware Australia Pty Ltd., 11 Endeavour Road, Caringbah, NSW 2229, Australia; Harriet Whiley 1,* , Jason Hinds 2, James Xi 2 and Richard Bentham 1 1 College of Science and Engineering, Flinders University, GPO Box 2100, Adelaide, SA 5001, Australia; Richard.Bentham@ﬂinders.edu.au 2 Enware Australia Pty Ltd., 11 Endeavour Road, Caringbah, NSW 2229, Australia; Jason.Hinds@enware.com.au (J.H.); James.Xi@enware.com.au (J.X.) * Correspondence: Harriet.Whiley@ﬂinders.edu.au; Tel.: +61-8-7221-8580 Harriet Whiley 1,* , Jason Hinds 2, James Xi 2 and Richard Bentham 1 1 College of Science and Engineering, Flinders University, GPO Box 2100, Adelaide, SA 5001, Australia; Richard Bentham@ﬂinders edu au 2 Enware Australia Pty Ltd., 11 Endeavour Road, Caringbah, NSW 2229, Australia; Jason.Hinds@enware.com.au (J.H.); James.Xi@enware.com.au (J.X.) * Correspondence: Harriet.Whiley@ﬂinders.edu.au; Tel.: +61-8-7221-8580 2 Enware Australia Pty Ltd., 11 Endeavour Road, Caringbah, NSW 2229, Australia; Jason.Hinds@enware.com.au (J.H.); James.Xi@enware.com.au (J.X.) * Correspondence: Harriet.Whiley@ﬂinders.edu.au; Tel.: +61-8-7221-8580 International Journal of Environmental Research and Public Health International Journal of Environmental Research and Public Health www.mdpi.com/journal/ijerph 1. Introduction Opportunistic premise plumbing pathogens (OPPPs) are an increasingly signiﬁcant public health issue [1–3]. In hospitals, it is argued that water distribution systems are the most important but overlooked source of hospital-acquired infections [3]. Faucets and water distribution systems have been recognised as reservoirs and sources of infection for a range of pathogens including Legionella spp., Pseudomonas aeruginosa, Aeromonas spp., Acinetobacter spp., Burkholderia spp., Enterobacter spp., Flavobacterium spp., Serratia marcescens, Stenotrophomonas maltophilia and non-tuberculous mycobacteria (NTM) [3–5]. Increasing aged populations and their associated elevated risk susceptibility are driving Int. J. Environ. Res. Public Health 2019, 16, 1332; doi:10.3390/ijerph16081332 www.mdpi.com/journal/ijerph 2 of 8 Int. J. Environ. Res. Public Health 2019, 16, 1332 the need for innovative approaches to monitoring and managing the risk from OPPPs in order to better protect public health [6]. An established source of OPPPs in hospital and healthcare water distribution systems is the municipal potable water supplies [7,8]. Once microorganisms enter a system, they form, or are incorporated into, bioﬁlm—a matrix of extracellular organic polymers combined with inorganic particles [4]. Bioﬁlm build-up and the corrosion of pipeline surfaces is a primary cause of decreased water quality [9]. Bioﬁlm formation also provides a nutrient source and protection from unfavourable environmental conditions [10]. Subsequent detachment of bioﬁlms provides a mechanism for further distribution, colonization, or transmission of pathogens, potentially resulting in human infection [11]. Numerous variables impact bioﬁlm formation, including water temperature, disinfection type and residual, pipe materials and sizes, as well as temporal changes in water hydraulics and chemistries [2]. One of the most signiﬁcant indicators of bioﬁlm formation is ﬂuctuating water usage and temporal stagnation [12–14]. Incorrectly balanced water hydraulics have been demonstrated to promote signiﬁcant bacterial growth [10]. A previous study of household drinking water found that overnight stagnation resulted in a 2–3-fold increase of cell concentrations [13]. Another study of an oﬃce building found that during the ﬁrst year of operation, stagnation resulted in signiﬁcant deterioration of water quality and increase in viable biomass [15]. The combined eﬀects of loss of temperature control and loosening of attached or suspended bioﬁlms can result in rapid removal and dissemination when ﬂow is re-established [12]. The prevention of stagnation in hot water systems has been recognised to reduce the risk of Legionella pneumophila in potable water distribution systems [9]. 1. Introduction In Australia, the delivery of hot water is regulated according to the National Building Code that captures the Australian Standard AS/NZS 3500. The standard sets required temperatures for storage and delivery of heated water to outlets. The standard also speciﬁes the temperature of water at outlets on the basis of the risk to occupants and their vulnerability. This standard encompasses both minimisation of microbial growth and the potential for scalding of users [16]. Installation of thermostatic mixing devices to deliver water at temperatures that minimise risk of scalding is an intrinsic part of the regulation. The necessity to deliver water at temperatures favourable to the survival and colonisation of OPPPs is an unavoidable risk of regulatory compliance. A global strategy in controlling this risk is to frequently ﬂush outlets to remove bioﬁlm debris and introduce residual disinfection [17]. Current regulations require the use of microbial water testing to inform the management and treatment of warm water systems. However, there are many limitations associated with the standard culture method of detection, including the underestimation of viable but non-culturable (VBNC) bacteria and the time delay from sampling to results. As such, there is a need for real-time monitoring strategies which will better inform risk management protocols [6]. This study investigated the novel approach of monitoring real-time temperature ﬂuctuations in thermostatic mixing valves (TMVs) located in an Australian hospital that were linked to ﬂow events and their relation to microbial water quality. The association with TMV average daily temperature, the number and duration of ﬂushing events on the day and up to seven days prior to sampling with concentrations of total heterotrophic bacteria and Legionella spp. were explored. 2.5. Statistics Statistical analysis was conducted using SPSS version 25.0 (IBM, NY, USA). A Kolmogorov– Smirnov test, Q-Q plot and histogram demonstrated that the data was not normally distributed. A linear mixed model was used to determine if there was a need to control for nesting of data due to tap location needs. The intraclass correlation coeﬃcient was 2.4%, therefore limited variability was due to TMV location and the microbial data was treated as independent. Scatterplots were created and the loess ﬁt line was used to visualise the association between variables. This was followed by calculating the non-parametric correlation coeﬃcient. A bootstrap solution for simple linear regression was used to examine the three-way association between the variables. The distribution of measurements was examined using a chi-squared goodness of ﬁt. This showed a bias in the distribution of measurement due to the higher frequency of sampling during November and December. However, due to the increase in the cold water temperature during these months (Australian summer), they pose the greatest risk for microbial growth. 2.3. Flushing Events As described above, water temperature data was continuously collected. An algorithm (see Supplementary Figure S1) was then used to identify and separate ﬂow events based on changes in temperature. These were broken into ﬂow/ﬂushing events greater or less than 15 s duration. The collected data was separated into diﬀerent categories according to the ﬂushing frequency. Total ﬂushing events on day of sampling, Total ﬂushing events less than 15 s duration on day of sampling, Total ﬂushing events longer than 15 s on day of sampling, Total ﬂushing events during the 3 days prior to sampling, Total ﬂushing events during the 7 days prior to sampling. 2.4. Microbiological Sampling A total of 865 water samples were collected from faucets attached to TMVs as part of the hospital’s mandatory veriﬁcation monitoring program in accordance with NSW Code of Practice for the Control of Legionnaires’ Disease (2004). Microbial analysis was conducted by a National Association of Testing Authorities Australia (NATA) accredited laboratory. Total heterotrophic bacteria were enumerated on plate count agar using AS/NZS 4276.3.1:2007; Legionella spp. (other than L. pneumophila), L. pneumophila SG1 and L. pneumophila serogroup 2–14 were enumerated on BCYE-GVPC agar in accordance with AS 3896:2008. 2.1. Study Site The study site was a hospital building less than 5 years old located in Sydney, NSW, containing 125 inpatient rooms spread over 12 ﬂoors. During the study period from 12 April 2013 until 22 May 2017, the temperature of water output from 220 TMVs was constantly monitored and 800 water samples were collected over the study period. The data presented in this study was collected as part of the hospital’s regulatory requirements and provides real world insight into the information used to manage water quality within hospital water distribution systems. 3 of 8 Int. J. Environ. Res. Public Health 2019, 16, 1332 2.2. Temperature The temperature of water outgoing from 220 TMVs located across the building was continuously monitored using a thermocouple temperature sensor hardwired back to an electronic water management system. The sensor was located just below the TMV outlet in a specially designed mixing chamber that ensured blending of the mixed water to improve temperature recording accuracy. This enabled every 0.5 ◦C temperature change to be recorded continuously. This was then reported as average daily temperature 7 days prior, 3 days prior and then on the day of sampling and microbial analysis. 3. Results The Spearman’s rho revealed a statistically significant relationship between the concentration of total heterotrophic bacteria and the number of flushing events seven days prior to sampling (rs[865] The Spearman’s rho revealed a statistically signiﬁcant relationship between the concentration of total heterotrophic bacteria and the number of ﬂushing events seven days prior to sampling (rs[865] = −0.188, p < 0.01) and three days prior to sampling (rs[865] = −0.151, p < 0.01), with increased ﬂow events associated with decreased concentrations of heterotrophic bacteria (Figure 2). The most signiﬁcant relationship was observed with the number of ﬂushing events during the 7 days prior to sampling. = −0.188, p < 0.01) and three days prior to sampling (rs[865] = −0.151, p < 0.01), with increased flow events associated with decreased concentrations of heterotrophic bacteria (Figure 2). The most significant relationship was observed with the number of flushing events during the 7 days prior to sampling. There was also a relationship between temperature and the total heterotrophic bacteria, with l i d i h hi h h hi b i (Fi 3) S ’ h There was also a relationship between temperature and the total heterotrophic bacteria, with lower temperatures associated with higher heterotrophic bacteria (Figure 3). Spearman’s rho revealed a statistically signiﬁcant association between total heterotrophic bacteria and average temperate on the day of sampling (rs[865] = −0.238, p < 0.01) and average temperature for the 7 days prior to sampling (rs[865] = −0.258, p < 0.01). lower temperatures associated with higher heterotrophic bacteria (Figure 3). Spearman’s rho revealed a statistically significant association between total heterotrophic bacteria and average temperate on the day of sampling (rs[865] = −0.238, p < 0.01) and average temperature for the 7 days prior to sampling (rs[865] = −0.258, p < 0.01). During the sampling period, only 4/865 (0.5%) samples were positive (limit of detection 10 colony forming units (CFU)/mL) for L pneumophila and all four were serogroup 1 No samples were During the sampling period, only 4/865 (0.5%) samples were positive (limit of detection 10 colony forming units (CFU)/mL) for L. pneumophila and all four were serogroup 1. No samples were positive for Legionella spp. other than L. pneumophila. All four water samples were also positive for total heterotrophic bacteria, and concentrations ranged from 690 CFU/mL to 14,000 CFU/mL. The number of ﬂow events and average temperatures for the sampling events which were positive for L. 3. Results A total of 629/865 (73%) of samples collected were positive for total heterotrophic bacteria at a range of 1–52,000. The temperature of the water at the sampling locations ranged from 25.0 ◦C to 89.0 ◦C, however, the median temperature was 25.7 ◦C. The number of ﬂow/ﬂushing events on the day of sampling ranged from 0–730 for events less than 15 s in duration, 0–639 for events greater than 15 s in duration and 0–1369 total events. 4 of 8 of 8 i i l Int. J. Environ. Res. Public Health 2019, 16, 1332 Int. J. Environ. Res. Public Health 2019, 16, x As the number of ﬂushing/ﬂow events on the day of sampling increased there was an initial increase in total heterotrophic bacteria observed. This could possibly be attributed to bioﬁlm detachment caused by sheering [18]. However, after ﬁve ﬂushing events there was a decline in total heterotrophic bacteria, which plateaued after around 10 ﬂushing events (Figure 1). The Spearman’s rho revealed that the relationship between the total number of ﬂushing event (rs[865] = −0.106, p < 0.01) and total heterotrophic bacteria was statistically signiﬁcant. This was also true for total number of ﬂushing events greater than 15 s (rs[865] = −0.126, p < 0.01), but not the total number of ﬂushing events less than 15 s (rs[865] = −0.017, p = 0.609). increase in total heterotrophic bacteria observed. This could possibly be attributed to biofilm detachment caused by sheering [18]. However, after five flushing events there was a decline in total heterotrophic bacteria, which plateaued after around 10 flushing events (Figure 1). The Spearman’s rho revealed that the relationship between the total number of flushing event (rs[865] = −0.106, p < 0.01) and total heterotrophic bacteria was statistically significant. This was also true for total number of flushing events greater than 15 s (rs[865] = −0.126, p < 0.01), but not the total number of flushing events less than 15 s (rs[865] = −0.017, p = 0.609). 3. Results pneumophila are shown in Table 1. The average temperature during the 7 days prior to sampling ranged from 25 ◦C to 30 ◦C. This temperature range was also associated with higher numbers of total heterotrophs. Two of the sampling sites had 11 and 17 ﬂushing events during the 7 days prior to sampling; however, sample site 45 had 136 and 139 ﬂushing events during the 7 days prior to sampling. colony forming units (CFU)/mL) for L. pneumophila and all four were serogroup 1. No samples were positive for Legionella spp. other than L. pneumophila. All four water samples were also positive for total heterotrophic bacteria, and concentrations ranged from 690 CFU/mL to 14,000 CFU/mL. The number of flow events and average temperatures for the sampling events which were positive for L. pneumophila are shown in Table 1. The average temperature during the 7 days prior to sampling ranged from 25 °C to 30 °C. This temperature range was also associated with higher numbers of total heterotrophs. Two of the sampling sites had 11 and 17 flushing events during the 7 days prior to sampling; however, sample site 45 had 136 and 139 flushing events during the 7 days prior to sampling. Figure 1. Simple scatter plot with loess line of fit showing the association between total heterotrophic bacteria (colony forming units (CFU)/mL) and total number of flushing events on the day of sampling. Figure 1. Simple scatter plot with loess line of ﬁt showing the association between total heterotrophic bacteria (colony forming units (CFU)/mL) and total number of ﬂushing events on the day of sampling. Figure 1. Simple scatter plot with loess line of fit showing the association between total heterotrophic bacteria (colony forming units (CFU)/mL) and total number of flushing events on the day of sampling. Figure 1. Simple scatter plot with loess line of ﬁt showing the association between total heterotrophic bacteria (colony forming units (CFU)/mL) and total number of ﬂushing events on the day of sampling. Figure 1. Simple scatter plot with loess line of fit showing the association between total heterotrophic bacteria (colony forming units (CFU)/mL) and total number of flushing events on the day of sampling. Figure 1. Simple scatter plot with loess line of ﬁt showing the association between total heterotrophic bacteria (colony forming units (CFU)/mL) and total number of ﬂushing events on the day of sampling. Int. J. Environ. Res. 3. Results Public Health 2019, 16, 1332 Int. J. Environ. Res. Public Health 2019, 16, x 5 of 8 5 of 8 Figure 2. Simple scatter plot with loess line of fit showing the association between total heterotrophic bacteria (CFU/mL) and the total number of flushing events during the 7 days prior to sampling. Figure 2. Simple scatter plot with loess line of ﬁt showing the association between total heterotrophic bacteria (CFU/mL) and the total number of ﬂushing events during the 7 days prior to sampling. Figure 2. Simple scatter plot with loess line of fit showing the association between total heterotrophic bacteria (CFU/mL) and the total number of flushing events during the 7 days prior to sampling. Figure 3. Simple scatter plot with loess line of fit showing the association between total heterotrophic bacteria (CFU/mL) and average water temperature (°C) during the 7 days prior to sampling. Figure 3. Simple scatter plot with loess line of ﬁt showing the association between total heterotrophic bacteria (CFU/mL) and average water temperature (◦C) during the 7 days prior to sampling. Fi 2 Si l tt l t ith l li f fit h i th i ti b t t t l h t t hi Figure 2. Simple scatter plot with loess line of ﬁt showing the association between total heterotrophic bacteria (CFU/mL) and the total number of ﬂushing events during the 7 days prior to sampling. Figure 2. Simple scatter plot with loess line of fit showing the association between total heterotrophic bacteria (CFU/mL) and the total number of flushing events during the 7 days prior to sampling. Figure 2. Simple scatter plot with loess line of ﬁt showing the association between total heterotrophic bacteria (CFU/mL) and the total number of ﬂushing events during the 7 days prior to sampling. Figure 2. Simple scatter plot with loess line of fit showing the association between total heterotrophic bacteria (CFU/mL) and the total number of flushing events during the 7 days prior to sampling. Figure 2. Simple scatter plot with loess line of ﬁt showing the association between total heterotrophic bacteria (CFU/mL) and the total number of ﬂushing events during the 7 days prior to sampling. Figure 2. Simple scatter plot with loess line of fit showing the association between total heterotrophic bacteria (CFU/mL) and the total number of flushing events during the 7 days prior to sampling. 3. Results g p p g p bacteria (CFU/mL) and the total number of flushing events during the 7 days prior to sampling. i l l h l l f f h h b l h h Figure 3. Simple scatter plot with loess line of fit showing the association between total heterotrophic bacteria (CFU/mL) and average water temperature (°C) during the 7 days prior to sampling. Figure 3. Simple scatter plot with loess line of ﬁt showing the association between total heterotrophic bacteria (CFU/mL) and average water temperature (◦C) during the 7 days prior to sampling. bacteria (CFU/mL) and the total number of flushing events during the 7 days prior to sampling. Figure 3. Simple scatter plot with loess line of fit showing the association between total heterotrophic bacteria (CFU/mL) and average water temperature (°C) during the 7 days prior to sampling. Figure 3. Simple scatter plot with loess line of ﬁt showing the association between total heterotrophic bacteria (CFU/mL) and average water temperature (◦C) during the 7 days prior to sampling. Figure 3. Simple scatter plot with loess line of fit showing the association between total heterotrophic bacteria (CFU/mL) and average water temperature (°C) during the 7 days prior to sampling. Figure 3. Simple scatter plot with loess line of ﬁt showing the association between total heterotrophic bacteria (CFU/mL) and average water temperature (◦C) during the 7 days prior to sampling. Figure 3. Simple scatter plot with loess line of fit showing the association between total heterotrophic bacteria (CFU/mL) and average water temperature (°C) during the 7 days prior to sampling. Figure 3. Simple scatter plot with loess line of ﬁt showing the association between total heterotrophic bacteria (CFU/mL) and average water temperature (◦C) during the 7 days prior to sampling. 6 of 8 Int. J. Environ. Res. Public Health 2019, 16, 1332 Table 1. Temperature, ﬂow events and total heterotrophic bacteria counts for samples which were positive for L. pneumophila serogroup 1. 4. Discussion This study presents a novel approach for monitoring building water quality using real-time surveillance of temperature and ﬂow events. This surveillance approach was validated by the statistically signiﬁcant association of increased heterotrophic bacteria observed at sampling points with few ﬂushing events and low temperatures. The association of increased heterotrophic bacteria with stagnant water is supported by previous research [13,14]. An investigation into bioﬁlm formation in a model distribution system found that increased stagnation promoted bioﬁlm formation. This relationship was also observed in a study of household faucets which found that overnight stagnation induced microbial growth [13]. Another study showed that the taps in homes that were very infrequently used had the highest bacterial contamination. Stagnation increased microbial numbers in water from those taps which were otherwise frequently used [14]. Although in this study there were not enough Legionella-positive samples for statistical analysis, previous studies have demonstrated Legionella concentrations to be positively associated with total heterotrophic bacteria at 22 ◦C and 37 ◦C, but not at temperatures above 55 ◦C [19]. Additionally, a study conducted in 1984 demonstrated that stagnant hot water storage tanks in a hospital were positive for Legionella and were seeding the distribution system. p p g g y This study also found that there was an association between increased ﬂushing events and increased water temperatures. In this hospital distribution system, the warm water ranged from 25.0 ◦C to 89.0 ◦C, and increased bacteria concentrations were associated with lower water temperatures. This is supported by a study of Quebec hospitals conducted in 1992 which demonstrated that increased Legionella concentrations were statistically signiﬁcantly (p = 0.03) associated with low water temperatures at faucet [20]. Additionally, a recent study using a model reticulating water system conﬁrmed that increased temperatures were critical for controlling L. pneumophila growth [21]. There is an observed relationship between the inﬂuences of ﬂushing events and temperatures at faucets. The ability to remotely monitor both of these factors that contribute to microbial colonisation and dissemination is of more relevance. This study validated that a remote monitoring strategy could be used to ensure good thermal control and minimisation of stagnation at outlets within potable water systems. The biggest advantage with the real-time monitoring strategy in this study is that it can be used to complement, and reduce the reliance on, microbial testing. 4. Discussion The detection of pathogens and other microbiological indicators is typically conducted oﬀsite, which is costly, time consuming and increases the risk of cross-contamination or loss of culturability [22,23]. 3. Results Sample Location ID Date Average Temperature (◦C) Number of Flushing Events Microbiological Results (CFU/mL) On the Day of Sampling During the 7 Days Prior to Sampling On the Day of Sampling During 7 Days Prior to Sampling Total Heterotrophic Bacteria Legionella pneumophila SG1 58 15 November 2013 26.9 25.4 10 11 690 10 45 31 January 2014 29.9 28.2 31 136 11,000 10 45 26 June 2014 31.1 29 36 139 14,000 20 63 25 September 2014 28 26.1 7 17 3500 20 Table 1. Temperature, ﬂow events and total heterotrophic bacteria counts for samples which were positive for L. pneumophila serogroup 1. Funding: This research received no external funding. Funding: This research received no external funding. Funding: This research received no external funding. Acknowledgments: Authors would like to thank Pawel Skuza, Flinders University, for his advice on the data analysis. Acknowledgments: Authors would like to thank Pawel Skuza, Flinders University, for his advice on the data analysis. Acknowledgments: Authors would like to thank Pawel Skuza, Flinders University, for his advice on data analysis. Conﬂicts of Interest: Harriet Whiley and Richard Bentham have no conﬂicts of interest to declare. Jason Hinds is the Research & Development Manager and James Xi and an electronics engineer for Enware Australia who provided the patented technology used in this study. References 1. Bentham, R.; Whiley, H. Quantitative Microbial Risk Assessment and Opportunist Waterborne Infections—Are There Too Many Gaps to Fill? Int. J. Environ. Res. Public Health 2018, 15, 1150. [CrossRef] 1. Bentham, R.; Whiley, H. Quantitative Microbial Risk Assessment and Opportunist Waterborne Infections—Are There Too Many Gaps to Fill? Int. J. Environ. Res. 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Public Health 2019, 16, 1332 Supplementary Materials: The following are available online at http://www.mdpi.com/1660-4601/16/8/1332/s1. Figure S1: Flow/ﬂushing event algorithm. Author Contributions: Conceptualization: R.B., J.H. and H.W.; methodology: H.W., J.H. and R.B.; software: J.X.; data curation: J.X.; analysis: H.W.; writing—original draft preparation: H.W.; writing—review and editing: H.W., R.B. and J.H. Author Contributions: Conceptualization: R.B., J.H. and H.W.; methodology: H.W., J.H. and R.B.; software: J.X.; data curation: J.X.; analysis: H.W.; writing—original draft preparation: H.W.; writing—review and editing: H.W., R.B. and J.H. References Role of stagnation and obstruction of water ﬂow in isolation of Legionella pneumophila from hospital plumbing. Appl. Environ. Microbiol. 1984, 48, 984–987. 10. Flemming, H.-C.; Wingender, J. The bioﬁlm matrix. Nat. Rev. Microbiol. 2010, 8, 623. [CrossRef] 11. Percival, S.; Walker, J. 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https://openalex.org/W2067067917	https://europepmc.org/articles/pmc3844277?pdf=render	English	null	A Review on Bioactivities of Perilla: Progress in Research on the Functions of Perilla as Medicine and Food	Evidence-based complementary and alternative medicine	2,013	cc-by	5,651	Hindawi Publishing Corporation Evidence-Based Complementary and Alternative Medicine Volume 2013, Article ID 925342, 7 pages http://dx.doi.org/10.1155/2013/925342 Hindawi Publishing Corporation Evidence-Based Complementary and Alternative Medicine Volume 2013, Article ID 925342, 7 pages http://dx.doi.org/10.1155/2013/925342 2. Methodology It is reported that Hangekobokuto can ameliorate sleep choking syndrome [3], swallowing reflex [4, 5], and panic disorder [6] in humans and has antidepressant effects in mice [7]. Ito et al. [8] reported that Kososan exhib- ited antidepressant-like effects in murine forced swimming model, and Suyu-Jiaonang (SYJN) demonstrated antistress effects in rats [9]. However, the importance and role of perilla in these Kampo medicines remain to be clarified. All published reports on physiological functions of perilla were extracted from the PubMed database. 1. Introduction medicines. In recent years, Kampo has attracted attention as an alternative medicine among the foreign countries, including those in Europe and the United States, and the World Health Organization (WHO) announced that they will add a chapter on traditional medicines, including Japanese Kampo, in the eleventh edition of the International Classi- fication of Diseases (ICD-11) in 2015. Accordingly, scientific investigations of the effects of Kampo are advancing [4– 9]. Nonetheless, for many years, Kampo has been used in traditional Asian medicines to treat physical conditions such as stress and asthma. Perilla is a type of aromatic vegetables. Asian people preferred consuming it since ancient times. It was used to treat fish and crab poisoning symptoms according to Chinese classics [1]. This information passed on from China throughout Asia; thereafter, perilla is used as a traditional medicine and functional food in Asia.t In traditional medicine, aromatic substances are often used to treat mental stress. Perilla is included as one of them, and it is also used in combination with other aromatic oriental medicines that are referred to as Kampo medicines (hereafter “Kampo”). Hangekobokuto (Chinese name: Banxia-Houpo- Tang), Kososan (Chinese name: Xiang-Su-San), and Suyu- Jiaonang (SYJN) are representative Kampo, and these are used to treat depression-related diseases and asthma [2]. We review the use of bioactive compounds from perilla varieties as medicines and foods to prevent and ameliorate illnesses. Miho Igarashi and Yoshifumi Miyazaki Center for Environment, Health and Field Sciences, Chiba University, Kashiwanoha 6-2-1, Kashiwa, Chiba 277-0882, Japan Center for Environment, Health and Field Sciences, Chiba University, Kashiwanoha 6-2-1, Kashiwa, Chiba 277-0 Correspondence should be addressed to Yoshifumi Miyazaki; ymiyazaki@faculty.chiba-u.jp Received 12 April 2013; Accepted 24 September 2013 Academic Editor: Wagner Vilegas Received 12 April 2013; Accepted 24 September 2013 Received 12 April 2013; Accepted 24 September 2013 Academic Editor: Wagner Vilegas Copyright © 2013 M. Igarashi and Y. Miyazaki. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Perilla is a useful pharmaceutical and food product and is empirically consumed by humans. However, its properties have not been evaluated extensively. In this review, we summarize the progress made in research, focusing on the bioactivities of perilla. There are many in vitro and animal studies on the cytostatic activity and antiallergic effects, respectively, of perilla and its constituents. However, its influence on humans remains unclear. Hence, investigating and clarifying the physiological effects of perilla and its constituents on humans are imperative in the future to adhere to the ideals of evidence-based medicine. 4. Bioactivity of Perilla 4.4. Effects of Perilla Oil. Perilla oil contains large quanti- ties of alpha-linolenic acid, an essential fatty acid, which decreases the risk of cardiovascular diseases [23]. Several in vitro, animal, and human nutritional studies have focused on the effects of perilla oil and have compared it with those of other oils. Perilla oil increases glucose-6-phosphatase activity, improves membrane stability [24], lowers plasma triacylglyc- erol levels [25], and controls liver fatty acid composition [26]. 4.4. Effects of Perilla Oil. Perilla oil contains large quanti- ties of alpha-linolenic acid, an essential fatty acid, which decreases the risk of cardiovascular diseases [23]. Several in vitro, animal, and human nutritional studies have focused on the effects of perilla oil and have compared it with those of other oils. Perilla oil increases glucose-6-phosphatase activity, improves membrane stability [24], lowers plasma triacylglyc- erol levels [25], and controls liver fatty acid composition [26]. 4.1. Effects of the Kampo Perillae Herba. Perillae herba has been used as an oriental medicine for many years in Asia and has been passed on through generations by experience. In Japan, evident effects of Kampo containing perillae herba have been recently reported. However, the effects of the perillae herba itself are not stated clearly. Perillae herba is included in Japanese Kampo medicines such as Hangekobokuto (Chinese name: Banxia Houpu; [3– 6, 12–15]), Kososan (Chinese name: Xiang-Su-San; [8]), and Saibokuto (Chinese name: Chai-Pu-Tang) for the treatment of cough and stress symptoms [2]. Perillae herba is also found in Chinese Suyu-Jiaonang (SYJN), which is also used to treat mental conditions [9, 16]. y p Perilla oil, which is rich in n-3 polyunsaturated fatty acids, regulates brown and white adipose tissue metabolism in a manner different from that of safflower and fish oils and contributes to physiological activities that prevent body fat accumulation, regulate glucose metabolism in rats [27], and control serum lipid concentrations [28]. Perilla oil rich in alpha-linolenic acid is effectively desaturated and elongated to form eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA, [29]). In addition, in light- and dark-discrimination learning tests, perilla oil-administrated senescence-accelerated mice (SAMP8) group exhibited higher discriminability than safflower oil-administrated group [30]. Moreover, serum lipids in the perilla oil- administrated SAMP8 group had a significantly greater ratio of apolipoprotein A-I (ApoA-I) to ApoA-II than those in the safflower-oil administered group [31]. Perilla oil was not 4.2. Effects of Perilla Decoctions. Antiallergic effects of perilla decoctions have been demonstrated on mice. 3. Perilla as Food and KAMPO Compounds Essential oil (−)-perillaldehyde, (+)-perillalcohol, (+)-limonene, alpha-pinene, and trans-shisool Pigment Cyanin and shisonin Other compounds Rosmarinic acid, adenine, and arginine 4.3. Effects of Perilla Extract. In cultured murine vascular smooth muscle cells, perilla extract has been shown to induce NO production, indicating that perilla may be useful for the prevention of vascular diseases such as arteriosclerosis [21]. In addition, perilla extract increased restraint and induced cell death in human hepatoma HepG2 cells [22]. Moreover, flow cytometry and DNA microarray experiments revealed significant apoptosis and time-dependent regulation of apoptotic genes, respectively, in cells treated with perilla leaf extracts. crispa), are commonly added to food in Japan. In addi- tion, perilla oil is extracted from Perilla ocymoides (Perilla frutescens (L.) Britton var. frutescens) for consumption in Asia [11] (Table 1). In Japanese pharmacy, leaves and branches of Perilla frutescence Britt. var. acuta Kudo and Perilla frutescence Britt. var. crispa Decaisne as Perillae Herba [10] (Table 1) are defined as medicines. Table 2 shows the main constituents of perillae herba [10]. However, in this study the experiment was conducted in vitro with a high dose of the perilla extract; therefore, it is unclear if perilla extract is effective in vivo. 3. Perilla as Food and KAMPO Two types of perilla, (Perilla frutescens (L.) Britton var. crispa (Thunb.) H. Deane f. purpurea (Makino) Makino and Perilla frutescens (L.) Britton var. crispa (Thunb.) H. Deane viridis i In Japan, these Kampo have been used as medicine for a long time and are now approved and used as general 2 Evidence-Based Complementary and Alternative Medicine 2 Table 1: Types of a perilla used for food and medicinal purposes [10, 11]. Scientific name Food Perilla frutescens (L.) Britton crispa (Thunb.) H. Deane f. purpurea (Makino) Makino Perilla frutescens (L.) Britton crispa (Thunb.) “Viridis crispa” Perilla frutescens (L.) frutescens Medicine Perilla frutescens Britton var. acuta Kudo Perillae Herba Perilla frutescens Britton crispa var. Decaisne Table 2: The main constituents in perillae herba [10]. Compounds Essential oil (−)-perillaldehyde, (+)-perillalcohol, (+)-limonene, alpha-pinene, and trans-shisool Pigment Cyanin and shisonin Other compounds Rosmarinic acid, adenine, and arginine Table 1: Types of a perilla used for food and medicinal purposes [10, 11]. Scientific name Food Perilla frutescens (L.) Britton crispa (Thunb.) H. Deane f. purpurea (Makino) Makino Perilla frutescens (L.) Britton crispa (Thunb.) “Viridis crispa” Perilla frutescens (L.) frutescens Medicine Perilla frutescens Britton var. acuta Kudo Perillae Herba Perilla frutescens Britton crispa var. Decaisne Table 2: The main constituents in perillae herba [10]. Compounds Essential oil (−)-perillaldehyde, (+)-perillalcohol, (+)-limonene, alpha-pinene, and trans-shisool Pigment Cyanin and shisonin Other compounds Rosmarinic acid, adenine, and arginine of the mucous membrane. Moreover, rosmarinic acid, present in high quantities in perilla, was found to be a constituent of perilla decoctions, and it is believed that the maximum effect of perilla decoction is caused by rosmarinic acid [17].hf p y The effects of rabbit anti-rat thymocyte serum were examined in a BALB/c mouse model of mesangioproliferative glomerulonephritis, and suppressive effects were observed [19].hf The positive effects of perilla decoctions were observed in ddY mice with type I allergies [18]. After the oral administra- tion of 500 mg/kg perilla decoction and the relative amount of rosmarinic acid in a mouse model with ear passive cuta- neous anaphylaxis (PCA), allergic reactions were inhibited. Because the inhibition rates of perilla decoctions and ros- marinic acid were approximately equal, it was believed that the effect of perilla decoction depended on rosmarinic acid [18, 20]. However, comparisons of the effects of rosmarinic acid with perilla decoctions indicate additional therapeutic constituents in perilla. Table 2: The main constituents in perillae herba [10]. 5. Effects of Perilla Constituents Perilla contains several essential oils, including (−)- perillaldehyde, (−)-perillylalcohol, (+)-limonene, alpha- pinene, and trans-shisool. Moreover, perilla contains the purple pigments shisonin and cyanin. Other constituents of perilla include rosmarinic acid, adenine, and arginine [10]. 5.2. Perillyl Alcohol. Perillyl alcohol is the subject of only few in vitro studies. In these studies, perillyl alcohol induced cell cycle arrest and cell death in BroTo; [38], and A549 [38]. Perillyl alcohol also had inhibitory effects on HCT116 cells (human colon cancer cell line; [44]) and caused dose- dependent inhibition of mammary tumor cell proliferation. 5.1. Effects of Perillaldehyde. In contrast with the Kampo cinnamaldehyde, perillaldehyde elicited antidepressant-like effects on the olfactory nervous system in mice that were subjected to chronic weak stress and a forced swimming test [33]. Antidepressant-like effects were evaluated by measuring the duration of immobility in the forced swimming test. Decreased duration of immobility was observed after 9 days of perillaldehyde inhalation. In contrast, the odorant cinnamaldehyde from cinnamon bark failed to decrease the duration of immobility. This antidepressant effect of perillaldehyde was abolished in mice induced with anosmia by zinc sulfate, confirming the olfactory mode of action. 5.3. Perillic Acid. Perillic acid has been shown to inhibit proliferation of HCT116 cells (human colon cancer, [44]), mammary tumor cells [45], and PC12 cells (rat pheochromo- cytoma; [39]), indicating anticancer properties. 5.4. Rosmarinic Acid. Rosmarinic acid is a constituent of rosemary; therefore, there are several reports on this con- stituent of perilla. 5.4. Rosmarinic Acid. Rosmarinic acid is a constituent of rosemary; therefore, there are several reports on this con- stituent of perilla. yi g y In Wistar rat aortas, vasodilatation following inhibition of Ca channels was ameliorated by perillaldehyde, with dose-dependent aorta extensibility, and relaxation with 0.01– 1 mM treatments with prostaglandin F2𝛼or norepinephrine [34]. In addition, these effects were unchanged by treatment with NG-nitro-L-arginine methyl ester or removal of aorta endothelium. Therefore, it was suggested that perillalde- hyde directly affects vascular smooth muscle. Furthermore, vasodilatation was not inhibited by the 𝛽-adrenergic-receptor blocker propranolol, the phosphodiesterase inhibitor theo- phylline, the delayed rectifier K+ channel blocker tetraethy- lammonium chloride, or the ATP-sensitive K+ channel blocker glibenclamide. In contrast, perillaldehyde caused vasodilatation of contracted aortas via a mechanism involv- ing Ca2+ transport out of the cell. Perillaldehyde caused slight vasodilatation of Ca2+-ionophore (A23187) contracted aortas but inhibited highly concentrated K+-mediated vasodilata- tion. 4. Bioactivity of Perilla In these studies, perilla decoctions partly controlled IgA nephropathy [17] and type I allergies [18]. It is thought that these effects are caused by rosmarinic acid. In addition, perilla decoctions demonstrated suppressive effects on mesangioproliferative glomerulonephritis in rats [18]. In HIGA mouse model (IgA renal damage), perilla decoctions alleviated IgA nephropathy through adjustments 3 Evidence-Based Complementary and Alternative Medicine antibacterial actions (using air washers) against floating microbes were reported [41]. as effective as soybean oil in preventing minor recurrent aphthous stomatitis; however, in vivo, in vitro, and lifestyle studies report positive effects of perilla oil on minor recurrent aphthous stomatitis [32]. Perillaldehyde was found to preserve fruits and promote the antioxidant activity of blueberries [42] and Chinese bayberries [43]. Hence, these natural products may be helpful in the maintenance of crop quality and security. However, further research is required to examine the influence of perillaldehyde on flavour, texture, and harvest parameters of crops. 5. Effects of Perilla Constituents Given the predominance of voltage-dependent Ca2+ efflux, it was suggested that perillaldehyde acted as a Ca2+ channel inhibitor for vasodilatation. It was reported that rosmarinic acid inhibits seasonal allergic rhinoconjunctivitis in humans [46]. This double- blind study indicated that oral supplementation with ros- marinic acid is an effective intervention for patients in the age group of 21–53 years with mild seasonal allergic rhinoconjunctivitis. In this 21-day experiment, rosmarinic acid significantly increased responder rates for itchy nose, watery eyes, itchy eyes, and total symptoms and decreased neutrophil and eosinophil numbers in nasal lavage fluid. The authors suggest that rosmarinic acid may reduce treatment costs for allergic diseases.h The absorption, metabolism, and urinary excretion of rosmarinic acid after a single dose of perilla extract were determined in six healthy men (mean age 37.2±6.2 years and mean body mass index 22.0 ± 1.9 kg/m2; [47]). In this study, rosmarinic acid was absorbed, conjugated, and methylated following intake, and a small proportion of rosmarinic acid was degraded into various constituents, including conjugated forms of caffeic acid, ferulic acid, and m-coumaric acid, which were rapidly excreted in urine.f In cultured human liver microsomes, perillaldehyde slightly inhibited the hydroxylation of bupropion (antide- pressant) by the enzyme CYP2B6 [35] and inhibited Candida albicans [36]. In addition, in 293 TRPA1-HEK cells, peril- laldehyde activated the transient potential A1 receptor [37], suggesting that this discovery will enable future analysis of taste properties. Several studies have examined the effects of rosmarinic acid on allergic reactions in mice. In nasal mucosa of ovalbumin-sensitized BALB/c mice, rosmarinic acid and 30% ethanol extract powder of perilla reduced the number of ear and eye rubs, the levels of IgE and histamine in the serum, inhibited protein and mRNA expression of interleukins (IL)-1𝛽, IL-6, and tumor necrosis factor-𝛼, and inhibited cyclooxygenase-2 protein expression and caspase-1 activity [48]. Moreover, rosmarinic acid blunted nuclear factor-kappa B (NF-𝜅B)/Rel A and caspase-1 activation. These results suggested that both rosmarinic acid and 30% ethanol extract powder of perilla alleviate allergic inflammatory reactions such as allergic rhinitis and allergic rhinoconjunctivitis. Perillaldehyde inhibited the proliferation of human squa- mous cell carcinoma of the tongue (BroTo) and human lung adenocarcinoma (A549) [38] and demonstrated anti- cancer activity in PC12 cells (rat pheochromocytoma cell line [39]). Moreover, Masutani et al. [40] reported the activation of the Nrf2-Keap1 system by perillaldehyde. Furthermore, Evidence-Based Complementary and Alternative Medicine 4 Table 3: The main effects of perilla. 5. Effects of Perilla Constituents Product studied Main effects Type of study Number of papers Perilla decoction Type I antiallergic effect Mouse ear passive cutaneous anaphylaxis (PCA) reaction IgA nephropathy in HIGA mice Decreased proliferation of mesangial cells Animal 5 Perilla extract Inhibits growth and induces NO production in vascular smooth muscle cells In vitro 2 Perilla oil Protective effects against recurrent aphthous stomatitis (−) Human 1 Fatty acid synthesis, improved learning in the Sidman active avoidance task Animal 8 Cytostatic activity In vitro 1 Perillaldehyde Antidepressant-like effect through regulation of the olfactory nervous system Animal 2 Vasodilatory effect Cytostatic activity and so on In vitro 6 Perillyl alcohol Cytostatic activity In vitro 2 Perillic acid Cytostatic activity In vitro 2 Rosmarinic acid Seasonal allergic rhinoconjunctivitis inhibition, absorption, metabolism, degradation, and urinary excretion Human 2 Antiallergic effects against type I allergies Antidepressant-like effect Anticarcinogenic effect Animal 8 Cytostatic activity In vitro 4 Caffeic acid Absorption, metabolism, degradation, and urinary excretion Human 1 Antidepressant-like effect Animal 1 Table 3: The main effects of perilla. cardiac muscle cells [55]. Moreover, Kim et al. suggested that rosmarinic acid should be viewed as a potential chemother- apeutic that inhibits cardiotoxicity in adriamycin-exposed patients. Indeed, rosmarinic acid significantly prevented 6- OHDA-induced cell viability reduction [56], indicating a potential as a chemotherapeutic agent for the treatment of Parkinson’s disease. Finally, rosmarinic acid inhibited the formation of reactive oxygen and nitrogen species in RAW264.7 macrophages [44]. Consistent with this report, rosmarinic acid ameliorated type I allergies in ddY mice [49] and inhibited ear-passive cutaneous anaphylaxis (PCA) reactions more effectively than the modern antiallergic drug tranilast. In HIGA mice that spontaneously develop high levels of serum immunoglobulin A (IgA) and mesangial IgA deposi- tion, rosmarinic acid suppressed serum IgA levels and may suppress IgA nephropathy [18]. In forced swimming tests, rosmarinic acid produced an antidepressant-like effect, at least in part via the proliferation of new-born cells in the dentate gyrus of the hippocampus [50]. Rosmarinic acid also reduced the defensive freezing behaviour of mice exposed to conditioned fear stress [51]. The authors suggest that investigating the mechanisms of these effects may help to explain the pathophysiology of such affective disorders, which would facilitate new therapeutic strategies and future development of novel anxiolytic and/or antidepressive drugs. 7. Discussion [2] “Notification of the medical devices investigation office, evalu- ation and licensing division,” in Ministry of Health LaWoJ, 2012. In this review, we were not able to find investigations on dietary perilla, though numerous studies revealed bioactive constituents of perilla. [3] A. Hisanaga, T. Itoh, Y. Hasegawa et al., “A case of sleep choking syndrome improved by the Kampo extract of Hange-koboku- to,” Psychiatry and Clinical Neurosciences, vol. 56, no. 3, pp. 325– 327, 2002. We searched the literature from medicine, pharmacology, and nutritional sciences to clarify the functions of perilla. We found very few studies of perilla on humans but many in vivo and in vitro investigations of its biological properties. Whereas dietary effects of perilla remain unknown, perilla oil is considered a high quality oil in nutritional sciences, which are concerned with lifestyle-related diseases. [4] K. Iwasaki, Q. Wang, H. Seki et al., “The effects of the traditional Chinese medicine, “Banxia Houpo Tang (Hange-Koboku To)” on the swallowing reflex in Parkinson’s disease,” Phytomedicine, vol. 7, no. 4, pp. 259–263, 2000. [5] K. Iwasaki, Q. Wang, T. Nakagawa, T. Suzuki, and H. Sasaki, “The Traditional Chinese Medicine Banxia Houpo Tang improves swallowing reflex,” Phytomedicine, vol. 6, no. 2, pp. 103–106, 1999. According to numerous in vivo and in vitro studies, perilla and its constituents have anticancer, antiallergy, and antide- pressant properties, and the biological activities of perilla and its constituents are increasingly well characterised (Table 3). Although there are several in vitro studies demonstrating anticancer properties, studies conducted in vivo are very few. Hence, further research is required to examine the effects of using perilla as a food and medicine in daily life. [6] N. Mantani, A. Hisanaga, T. Kogure, T. Kita, Y. Shimada, and K. Terasawa, “Four cases of panic disorder successfully treated with Kampo (Japanese herbal) medicines: kami-shoyo-san and Hange-koboku-to,” Psychiatry and Clinical Neurosciences, vol. 56, no. 6, pp. 617–620, 2002. [7] L. Luo, J. Nong Wang, L. D. Kong, Q. G. Jiang, and R. X. Tan, “Antidepressant effects of Banxia Houpu decoction, a traditional Chinese medicinal empirical formula,” Journal of Ethnopharmacology, vol. 73, no. 1-2, pp. 277–281, 2000. Among active constituents of perilla, perillaldehyde and rosmarinic acid have received the most attention, suggesting that these compounds are important constituents. Perillalde- hyde contributes to aroma and accounts for approximately 50% of the oil refined from perillas. Evidence-Based Complementary and Alternative Medicine Evidence-Based Complementary and Alternative Medicine The authors declare no conflict of interests. The authors declare no conflict of interests. 8. Conclusion Animal and in vitro studies abundantly demonstrate the effects of perilla and its constituents; however, their influence on humans is nearly unknown. In contrast, perilla has already been used as a form of oriental medicine and as food. Investigating and clarifying the physiological effects of perilla and its constituents on humans are imperative in the future to adhere to the ideals of EBM. [14] T. Naito, H. Itoh, and M. Takeyama, “Effects of Hange-koboku- to (Banxia-houpo-tang) on neuropeptide levels in human plasma and saliva,” Biological and Pharmaceutical Bulletin, vol. 26, no. 11, pp. 1609–1613, 2003. [15] T. Naito, H. Itoh, and M. Takeyama, “Some gastrointestinal function regulatory Kampo medicines have modulatory effects on human plasma adrenocorticotropic hormone and cortisol levels with continual stress exposure,” Biological and Pharma- ceutical Bulletin, vol. 26, no. 1, pp. 101–104, 2003. 7. Discussion Therefore, it is considered important to examine the effects of this compound on stress; as for Kampo and food, its pleasant aroma is well known. However, only one paper considers bioactivity from aroma alone [31]. As rosmarinic acid is a constituent of both perilla and rosemary, scientific data is available for rosmarinic acid than for the other perilla compounds. Consequently, its effects on allergies are well characterised in humans [46]. [8] N. Ito, T. Nagai, T. Yabe, S. Nunome, T. Hanawa, and H. Yamada, “Antidepressant-like activity of a Kampo (Japanese herbal) medicine, Koso-san (Xiang-Su-San), and its mode of action via the hypothalamic-pituitary-adrenal axis,” Phytomedicine, vol. 13, no. 9-10, pp. 658–667, 2006. [9] Q.-Q. Mao, Z. Huang, X.-M. Zhong et al., “Effects of SYJN, a Chinese herbal formula, on chronic unpredictable stress- induced changes in behavior and brain BDNF in rats,” Journal of Ethnopharmacology, vol. 128, no. 2, pp. 336–341, 2010. [10] I. Kitagawa, J. Kinjyo, H. Kuwasima et al., Pharmacognosy, Hirokawa, Tokyo, Japan, 7th edition, 2011. f In this review, we have summarized the current progress in perilla research. Although we found abundant in vitro and animal studies, we could not find a human study regarding the traditional theory on the effects of Kampo aroma on stress and depression. Investigating and clarifying the physiological effects of perilla and its constituents on humans are imperative in the future to adhere to the ideals of evidence-based medicine (EBM). [11] T. Makino, New Makino Japan Plant, Diagram Model Hokuryukan, Tokyo, Japan, 2008. [12] A. Kaneko, S. Cho, K. Hirai et al., “Hange-koboku-to, a kampo medicine, modulates cerebral levels of 5-HT (5- hydroxytryptamine), NA (noradrenaline) and DA (dopamine) in mice,” Phytotherapy Research, vol. 19, no. 6, pp. 491–495, 2005. [13] J.-M. Li, L.-D. Kong, Y.-M. Wang, C. H. K. Cheng, W.-Y. Zhang, and W.-Z. Tan, “Behavioral and biochemical studies on chronic mild stress models in rats treated with a Chinese traditional prescription Banxia-houpu decoction,” Life Sciences, vol. 74, no. 1, pp. 55–73, 2003. Conflict of Interests [16] Q.-Q. Mao, X.-M. Zhong, Z.-Y. Li, C.-R. Feng, A.-J. Pan, and Z. Huang, “Herbal formula SYJN increases neurotrophin-3 and nerve growth factor expression in brain regions of rats exposed to chronic unpredictable stress,” Journal of Ethnopharmacology, vol. 131, no. 1, pp. 182–186, 2010. 6. Other Constituents Perillaketone has been shown to activate cloned TRPA1 channels in vitro [37]. Bassoli et al. [37] suggested that these data may explain the taste properties of the plant at the molecular level and identify it as an interesting target for its culinary and pharmaceutical applications. In other studies, rosmarinic acid demonstrated anticar- cinogenic effects in a murine two-stage skin model [52]. This paper concluded that rosmarinic acid contributes to the anticarcinogenic effects of perilla via two independent mech- anisms: by inhibiting inflammatory responses and potent superoxide scavenging activity. Furthermore, rosmarinic acid ameliorated LPS-induced liver injury in D-GalN-sensitized mice [53]. Carvane was shown to inhibit the transformation of Candida albicans [36] and also inhibited type I allergies in ddY mice [18]. Luteolin inhibits inflammation and allergic responses in vivo and in vitro [57], and the mechanisms of relaxant action were elucidated in isolated guinea pig tracheas [58].h The perilla constituent 1,2-Di-O-alpha-linolenoyl-sn- glycerol, which was extracted from the local variety, kida- chirimen shiso, inhibited superoxide generation [59]. In vitro, rosmarinic acid inhibited mesangial-cell pro- liferation [54] and adriamycin-induced apoptosis in H9c2 5 [1] Y. Takahashi, Handbook of Modern Chinese Medicine II, Yakkyoku Shimbun, Tokyo, Japan, 1969, [Japanese]. References [1] Y. Takahashi, Handbook of Modern Chinese Medicine II, Yakkyoku Shimbun, Tokyo, Japan, 1969, [Japanese]. [17] T. Makino, T. Ono, K. 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https://openalex.org/W4287198866	https://zenodo.org/records/6392427/files/2121mechatroj04.pdf	English	null	MECHATRONIC CAPSTONE DESIGN COURSE	Zenodo (CERN European Organization for Nuclear Research)	2,021	cc-by	6,172	Mechatronics and Applications: An International Journal (MECHATROJ), Vol. 2, No.1 Mechatronics and Applications: An International Journal (MECHATROJ), Vol. 2, No.1 ABSTRACT Mechatronic system design is multidisciplinary and integration among the mechanical, sensor, actuator, electronic, computer, and control elements is essential. The integration is done simultaneously from the beginning and the design is model-based. Modeling, physical and mathematical, is the key in modern engineering practice. Before any hardware is purchased or built, a complete, computer virtual prototype is created to meet all performance specifications. Only then can an actual hardware prototype be built. Mechatronics students need to perform, need to experience, this process before graduation. It is all about the process, as they will be asked to apply the process to challenging problems. Modern machines are complex and computer-controlled, but most are made up of mechanisms. In this course, senior mechanical engineering students choose a mechanism to perform a certain task. They then apply the process to create the virtual and hardware prototypes to meet performance specifications, just like real mechatronics engineers. MECHATRONIC CAPSTONE DESIGN COURSE Kevin Craig Department of Engineering, Hofstra University, Hempstead, NY, USA KEYWORDS Mechatronic Design Process, Mechatronic System Design, Mechatronics Education, Senior Capstone Design, Virtual and Hardware Prototypes 1. INTRODUCTION Mechatronics is the best practice by engineers driven by the needs of industry and human beings. It is technology integration to achieve optimal system functionality, the synergistic integration of physical systems, electronics, controls, and computers through the design process, from the very start of the design process, thus enabling complex decision making. Integration is the key element in mechatronic design as complexity has been transferred from the mechanical domain to the electronic and computer software domains. Mechatronics is an evolutionary design development that demands horizontal integration among the various engineering disciplines, as well as vertical integration between design and manufacturing, and is what modern mechanical engineering needs to be. 47 47 Mechatronics and Applications: An International Journal (MECHATROJ), Vol. 2, No.1 As a professor at Rensselaer Polytechnic Institute from 1989-2008, I created the mechatronics undergraduate and graduate programs there, and, in 1995, created the Mechatronics diagram (Figure 1), now used around the world to illustrate this multidisciplinary field. Design, above all else, defines the difference between an engineering education and a science education. Design represents the bridge between theory and reality. It is the process by which our ideas enter and influence the world around us. Design distinguishes us as engineers. This one-semester, three-credit senior capstone design course for mechanical engineers is intended to be a challenging multidisciplinary design experience prior to graduation. This course builds upon the knowledge and skills that students have gained from other engineering courses taken as part of the mechanical engineering curriculum, in particular, the junior-level, four-credit required courses Modeling, Analysis, & Control of Dynamic Systems, Mechanical Engineering Design, and Mechatronic System Design. It provides a meaningful culminating experience that introduces students to the multidisciplinary, mechatronic aspects of design and to the essential model-based approach to design, rather than the design-build-test approach that is so common in a senior capstone design course. Under the guidance of the instructor, students develop an appreciation for the depth and breadth of knowledge and skills necessary for successful implementation of a significant development effort. The integrative and iterative aspects of a capstone design experience are emphasized. Students are required to apply their skills in multidisciplinary design, modeling, analysis, simulation, verification, and computer-control design, with electronics, sensors, actuators, microcontrollers, and real-time programming. Professional development in areas of team dynamics, interpersonal relationships, and technical communications are learned via active participation. All mechanical engineering programs have a senior capstone design course. • The course is either a one-semester, three-credit course or a two-semester, four-credit to six-credit course. 1. INTRODUCTION It is an ABET (Accreditation Board for Engineering and Technology) requirement. A review of senior mechanical engineering capstone courses around the country will uncover considerable differences. Among the differences are: • The course is either a one-semester, three-credit course or a two-semester, four-credit to six-credit course. 48 Mechatronics and Applications: An International Journal (MECHATROJ), Vol. 2, No.1 • The design project is either purely mechanical or is multidisciplinary, with sensor, actuator and micro-computer control. • The design project is either purely mechanical or is multidisciplinary, with sensor, actuator and micro-computer control. p e design project is industry-sponsored or is proposed by the student design team. p • The design project is industry-sponsored or is proposed by the student design team. Funding ranges from industry financial support up to several thousand dollars, to $400 - $ • Funding ranges from industry financial support up to several thousand dollars, to $1000 per 4-person team funded by the mechanical engineering department. p p y g g p • The project can be virtual, i.e., a paper design and slide presentation are the end result, with or without a working virtual prototype. Or the project can include both a complete working model-based virtual prototype, followed by a working hardware prototype. • The project can be virtual, i.e., a paper design and slide presentation are the end result, with or without a working virtual prototype. Or the project can include both a complete working model-based virtual prototype, followed by a working hardware prototype. g p yp y g p yp • The approach used is design-build-test, with little or no physical / mathematical modeling, or the design must consist of a model-based virtual prototype, either alone, or followed by a working hardware prototype. • The approach used is design-build-test, with little or no physical / mathematical modeling, or the design must consist of a model-based virtual prototype, either alone, or followed by a working hardware prototype. • The design experience can be run as a formal class, with 3-4 person teams, or as an independent study activity with 3-4 person teams or individual projects. • The design experience can be run as a formal class, with 3-4 person teams, or as an independent study activity with 3-4 person teams or individual projects. 2. MECHATRONIC MACHINE DESIGN A modern mechatronic machine is like the human body. The actuators are the muscles that make things happen. The sensors are the senses that tell us what is happening. The links and joints of the mechanisms are the legs, arms, hands, and joints of the human body, and the microcontroller, which performs the complex decision making it has been programmed to do, is the human brain. Mechanisms have been around for millennia dating back to the Egyptians. Up until 30 years ago, the design of mechanisms was purely mechanical, often with mechanical cams, but, in the present mechatronic age, the design is multidisciplinary, i.e., mechanical, electrical, electro- mechanical, hydraulic, and pneumatic, all computer-controlled, with electronic cams and complex motion profiles (See Figure 2). What does it mean to mechatronify a mechanism or machine [1]? In life, how you do something is more important than what you do. Mechatronics is more than just adding a sensor, an actuator, and a computer control system. They must be added in an integrated way from the very start of the design and, just as importantly, using a model-based design process that takes advantage of analysis techniques and simulation software and leads to optimum designs without trial and error. Combining old inventions with new technology fosters innovative ideas, but it is the process that transforms these ideas into reality. What does it mean to mechatronify a mechanism or machine [1]? In life, how you do something is more important than what you do. Mechatronics is more than just adding a sensor, an actuator, and a computer control system. They must be added in an integrated way from the very start of the design and, just as importantly, using a model-based design process that takes advantage of analysis techniques and simulation software and leads to optimum designs without trial and error. Combining old inventions with new technology fosters innovative ideas, but it is the process that transforms these ideas into reality. What does it mean to mechatronify a mechanism or machine [1]? In life, how you do something is more important than what you do. Mechatronics is more than just adding a sensor, an actuator, and a computer control system. 2. MECHATRONIC MACHINE DESIGN They must be added in an integrated way from the very start of the design and, just as importantly, using a model-based design process that takes advantage of analysis techniques and simulation software and leads to optimum designs without trial and error. Combining old inventions with new technology fosters innovative ideas, but it is the process that transforms these ideas into reality. A mechatronic approach to the design and implementation of any mechanism has been developed that reflects both the traditional mechanism analysis and synthesis methods together with the best industry practices, e.g., Rockwell Automation, Procter & Gamble. It is shown in the flow chart 49 Mechatronics and Applications: An International Journal (MECHATROJ), Vol. 2, No.1 (Figure 3) [2]. It is through this process that innovative ideas become a reality. Sweating the details with a combination of knowledge, old and new, process, and determination will make innovation happen. System requirements dictate a desired end-point trajectory. The motion can be defined as an electronic cam, characterized by different profiles and maximum values of velocity, acceleration, and jerk, which will affect the level of mechanical stress, vibration, and noise in the motor, transmission system, and mechanical load. It is essential that the desired motion profile be chosen first because the required torque vs. speed curve to size the motor depends on it. In addition, the motion profile has relevant implications on the tracking errors through the control system. A kinematic (geometry of motion) model of the mechanical system is then developed, and through inverse kinematics, the required motor motion profile is determined. The torque- speed requirements for the motor are determined by first developing a kinetic (geometry plus all torques and mass moments of inertia) model of the complete mechanical system and then applying an appropriate feedback control system to that model. A computer simulation (e.g., MatLab Simulink) of the mechanical and control systems will result in the necessary torque- speed curve of the load to size the motor. Candidate servo motors (e.g., brushless DC motor) can now be identified. Additional requirements, e.g., cost, energy efficiency, and load-to-motor inertia ratio, will shorten the list. The chosen motor, including any flexible couplings or gearing, becomes an integral part of the system and its properties must be included in the system model. The control system will have to be tuned or even modified because of the motor addition. 2. MECHATRONIC MACHINE DESIGN A computer simulation will reveal new torque-speed requirements for the system. Is the motor’s torque-speed capability satisfactory? Is the control system stable? Does the system meet application-specific requirements regarding time response, relative stability, and steady-state error? If the answer to any of these questions is no, iteration is required. A model-based design approach, together with computer simulation, will lead to an optimal motor selection with all the benefits that implies. 50 Mechatronics and Applications: An International Journal (MECHATROJ), Vol. 2, No.1 3. SLIDER-CRANK CASE STUDY Let’s use as an example a mechanism developed by Leonardo da Vinci over 500 years ago and now found in engines, automation applications, and miniature devices around the world – the slider crank. Let’s illustrate how to mechatronify this Renaissance mechanism. Shown is a diagram of a slider-crank mechanism (Figure 4), consisting of a flywheel-crank, a connecting rod, and a slider, all assumed to be rigid. The external forces / torques acting are the servo-motor torque τ (and also motor friction torque, not shown), the slider friction force Ff, and the external force Fe. It is a special case of the four-bar linkage where one crank is infinite in length, such that its end point (point B) has rectilinear motion. It is a one-degree-of-freedom system, as only one coordinate is needed to completely describe its motion. The constraint equation relating angles θ and φ is r•(sin θ) = ℓ•(sin φ). Kinematic analysis (i.e., the geometry of motion) can be carried out graphically yielding great insight. However, a mathematical solution is much more effective for mechatronic system design and optimization. This analysis can be performed either by trigonometry or by complex numbers. Positions, velocities, and accelerations of key points are obtained, as well as the angular velocities and angular accelerations of the rigid bodies. Kinetic analysis can be accomplished by drawing free-body diagrams showing gravitational forces, contact forces / torques, and also the inertia forces / torques, and then summing forces / moments, as needed. This is known as the D’Alembert approach to applying the Newton-Euler Equations, and forces / torques at all joints can be determined. 3. SLIDER-CRANK CASE STUDY The system equation of motion is directly obtained by the application of y q y y pp Lagrange’s Equation d T T V Q dt           using the generalized coordinate θ, where V is the system potential energy, i.e., 1 2 2 V m grsin  , T is the system kinetic energy, i.e.,   2 2 2 2 1 1 1 1 1 2 2 2 3 3 2 2 2 2 d d T I I m v + m v dt dt                                  , and Qθ is the generalized torque due to torques that do work, i.e.,     r e f c Q F F rsin 1 cos     with 2 2 2 c r sin   . The resulting equation has the form   2 2 d d M N , F dt dt              .   2 2 2 2 1 1 1 1 1 2 2 2 3 3 2 2 2 2 d d T I I m v + m v dt dt                                  and Qθ is the generalized torque due         , and Qθ is the generalized torque due to torques that do work, i.e.,     r e f c Q F F rsin 1 cos     with 2 2 2 c r sin   . The resulting equation has the form   2 2 d d M N , F dt dt              . resulting equation has the form 51 Once the kinematic and kinetic analyses are completed, the desired end-point trajectory must be defined, and then, through inverse kinematics, which includes here the crank and connecting rod lengths, the necessary motion profile for the actuator is computed. 3. SLIDER-CRANK CASE STUDY This is accomplished by 51 51 Mechatronics and Applications: An International Journal (MECHATROJ), Vol. 2, No.1 Mechatronics and Applications: An International Journal (MECHATROJ), Vol. 2, No.1 trajectory planning. This profile needs to be defined in a way to avoid or reduce the mechanical vibration and stress on components and actuators, as well as to reduce overshoot response and excessive position error during motion. This is accomplished by electronic cams. The inverse kinetic analysis, which includes masses, center-of-mass locations, and mass moments of inertia, is used to generate the required actuator torque / force for the motion profile, and results in a speed / torque-force diagram on which to base actuator selection. The chosen actuator now becomes part of the system, and, with the updated system, a control system, with feedback and feedforward control, is designed, which then results in a new speed / torque-force profile. The entire system should now be simulated, with the addition of any parasitic effects, for design validation. To create a mechatronic machine requires an integrated approach and a process that results in a complete virtual prototype before any work is done to create the actual working prototype. Once the virtual prototype – complete with models of the mechanical elements, electronics, controller, microcomputer, sensors, and actuators – is shown to work as desired, then work towards building the actual prototype can begin. When completed, the working prototype should work as expected, the first time power is turned on. That is called model-based design and it is the cornerstone of modern engineering practice. 4. SENIOR CAPSTONE DESIGN COURSE The mechatronic mechanism design process was implemented, and a slider crank was built to accomplish a prescribed task. This process is essential to modern engineering practice and this case study was used as a guide in Mechanical Engineering Senior Capstone Design during the fall 2018 semester. The four-bar linkage is probably the most common mechanism in the world and is used in a great variety of applications. Twenty-eight senior ME students, working in teams of 4, were assigned a four-bar mechanism application: aircraft landing gear, quick-return mechanism, pick-and-place mechanism, robot gripper, straight-line mechanism, flipping mechanism, and windshield wiper, and then proceeded through a process to create a virtual prototype and then, a working prototype. Pictures appear in the Appendix. Table 1 Course Milestones Due Dates Form Teams & Develop Challenge Concept Week #1 Virtual Prototype Demonstration and Presentation Week #7 Working Prototype Demonstration and Presentation Week #14 Final Presentation with Virtual & Working Prototypes and Design Notebook Exam Week The course learning objectives, major deliverables, course organization, course policies, grade summary, and weekly team tasks are shown below. Process Steps:  Define project specifications. These are continually reviewed and updated.  Create the motion profile.  Define project specifications. These are continually reviewed and updated.  Model the mechanism forward and inverse kinematics.  Model the inverse kinetic system to determine the speed-torque requirements and select a motor.  Model the forward kinetics with the selected motor included in the model.  Design a feedback control system to perform the desired motion.  Create and simulate the complete virtual prototype with mechanics, sensors, motor, electronics, and controller included.  Once satisfied with the performance of the virtual prototype, work on the actual working prototype can begin using fundamental machine-design principles.  When the actual working prototype is completed, its performance is compared to t predications from the virtual prototype. Discrepancies are resolved. The motors used were brushed dc motors (with and without a gear box) equipped with optical encoders. The controller was designed in MatLab Simulink, tested on the Simulink nonlinear The motors used were brushed dc motors (with and without a gear box) equipped with optical encoders. The controller was designed in MatLab Simulink, tested on the Simulink nonlinear 52 Mechatronics and Applications: An International Journal (MECHATROJ), Vol. 2, No.1 virtual prototype, and then implemented on the working prototype using a H-bridge and Lab VIEW with the NI myRIO controller (Figure 5). The students experience what they will actually be asked to do as practicing engineers. Students maintain a bound design notebook, one per team, in which all information, including minutes from meetings, reference material gathered, and all technical work performed. If it is not in the notebook, from the instructor’s point of view, it does not exist. The course milestones are shown in Table 1. Course Organization  The course consisted of two mandatory 85-minute sessions each week for all students. The first session each week was spent in a classroom setting during which the instructor covered fundamental engineering content essential for successful completion of the challenge. In the second session each week, each team worked together and with the instructor, as well as discussed and presented their work in a mini-design review format to the other teams.  Students were organized into four-person multidisciplinary teams during the first period of the course.  During the first period, each team was assigned a four-bar mechanism design challenge. The system necessarily was dynamic with sensors, electronics, actuators, and computer control as integral parts of the design, i.e., a mechatronic system. Major Deliverables 1. The engineering details of a concept design (physical and mathematical modeling, model analysis and verification, virtual prototype, computer-aided design drawings, electrical schematics, performance test results) as described in an engineering notebook with a presentation. 1. The engineering details of a concept design (physical and mathematical modeling, model analysis and verification, virtual prototype, computer-aided design drawings, electrical schematics, performance test results) as described in an engineering notebook with a presentation. 2. A working prototype which the team members design, fabricate, test, and demonstrate at a design exposition. Course Learning Objectives: 1. To provide the student with an experience that requires active student participation. 2. To enhance the student’s skills in engineering design methodology, including research, modeling, analysis, computer simulation, virtual and real prototype testing, and participation in design reviews. 3. To simulate the advanced product development process used in industry including the incorporation of customer requirements, performing a state-of-the-art search, keeping an engineering notebook, and organizing a commercial presentation of the work. g g g g p 4. To illustrate the interaction between competing technical and non-technical issues and the role of compromise, constraints and the interplay of potential benefits versus risks. 5. To provide the student with exposure to various phases of the design process, from the specification of requirements and constraints to product realization. All phases of new product development are practiced: concept formulation, technology search, preliminary design and layout, virtual prototyping, detailed design, fabrication, parts procurement, assembly, testing, and documentation. 53 Mechatronics and Applications: An International Journal (MECHATROJ), Vol. 2, No.1 Mechatronics and Applications: An International Journal (MECHATROJ), Vol. 2, No.1 6. To help develop an understanding of the planning, coordination, cooperation, and communication required in a team effort. 6. To help develop an understanding of the planning, coordination, cooperation, and communication required in a team effort. 7. To have the student understand what is required to meet a firm technical deadline where funds and technical assistance are limited: scheduling work, developing contingencies, specifying, procuring and incorporating purchased parts, identifying and using available fabrication and test equipment. 8. To allow for innovation. 8. To allow for innovation. 9. To allow the student to apply the skills learned in previous engineering courses to a challenging project. 9. To allow the student to apply the skills learned in previous engineering courses to a challenging project. Class Attendance, Participation, Preparation, and Conduct  Design is not a spectator sport. Active participation is required for a meaningful capstone experience. Students were expected to attend and participate in all class sessions and make relevant contributions in team meetings outside of regularly scheduled class time. Active participation and initiative were critical parts of the student’s individual success and that of the team.  Design is not a spectator sport. Active participation is required for a meaningful capstone experience. Students were expected to attend and participate in all class sessions and make relevant contributions in team meetings outside of regularly scheduled class time. Active participation and initiative were critical parts of the student’s individual success and that of the team.  Attendance at all classes was mandatory.  St dent participation in class as strongl enco raged  Attendance at all classes was mandatory.  Student participation in class was strongly encouraged.  Preparation for class was essential.  All course notes / announcements were posted on the course web site.  Students were expected to conduct themselves in a professional manner at all times with integrity, honesty, and respect for others.  Students were expected to conduct themselves in a professional manner at all times with integrity, honesty, and respect for others. Grade Summary 2, No.1 Design Notebook 20% Total 100% Weekly Team Tasks Table 2 Team Tasks Week # Form 4-Person Teams; Select & Research 4-Bar Mechanism Task; Set Up Design Notebook Week #1 Define Mechanism Performance Requirements & Motion Profile for the Selected 4-Bar Mechanism Task; Perform Forward and Inverse Kinematic Analysis & Implement in Simulink Week #2 Perform Forward & Inverse Kinetic Analysis using Newton-Euler Approach; Determine Equation of Motion using the Lagrange Approach; Implement in Simulink Week #3 Determine Motor Speed-Torque Requirement; Identify Candidate Brushed DC Motors; Choose Motor & Justify Week #4 Augment System with Motor (Inertia and Friction); Design Controller using MatLab; Implement Closed-Loop System in Simulink; Reevaluate Motor Selection Week #5 Complete the Virtual Prototype Design & Prepare Presentation & Report; Order Motor Week #6 Virtual Prototype Presentation and Report Week #7 Develop Mechanism Detailed Design using Machine Design Principles Week #8 Detailed Design & Build Week #9 Detailed Design & Build Week #10 Detailed Design & Build Week #11 H-Bridge Set-Up; LabVIEW Real-Time Programming; System Closed-Loop Testing Week #12 Complete the Actual Prototype Build & Test; Prepare Presentation & Report Week #13 Actual Prototype Presentation and Report Week #14 Team Tasks Week # Form 4-Person Teams; Select & Research 4-Bar Mechanism Task; Set Up Design Notebook Week #1 Define Mechanism Performance Requirements & Motion Profile for the Selected 4-Bar Mechanism Task; Perform Forward and Inverse Kinematic Analysis & Implement in Simulink Week #2 Perform Forward & Inverse Kinetic Analysis using Newton-Euler Approach; Determine Equation of Motion using the Lagrange Approach; Implement in Simulink Week #3 Determine Motor Speed-Torque Requirement; Identify Candidate Brushed DC Motors; Choose Motor & Justify Week #4 Augment System with Motor (Inertia and Friction); Design Controller using MatLab; Implement Closed-Loop System in Simulink; Reevaluate Motor Selection Week #5 Complete the Virtual Prototype Design & Prepare Presentation & Report; Order Motor Week #6 Virtual Prototype Presentation and Report Week #7 Develop Mechanism Detailed Design using Machine Design Principles Week #8 Detailed Design & Build Week #9 Detailed Design & Build Week #10 Detailed Design & Build Week #11 H-Bridge Set-Up; LabVIEW Real-Time Programming; System Closed-Loop Testing Week #12 Complete the Actual Prototype Build & Test; Prepare Presentation & Report Week #13 Actual Prototype Presentation and Report Week #14 Course Evaluation Twenty-seven students evaluated the course. (Questionnaire and results are in the Appendix). The questionnaire had an overall quality question plus three parts. In Part A, students rated their ability to apply knowledge and tools required for industry; in Part B, students rated which skills were most useful for a practicing engineer; and in Part C, students ranked how the course prepared them for the future. Parts A and C applied specifically to the course. A rating of one meant the course strongly achieved the goal, while a five meant the course did not. The overall course quality was rated 1.63. Students strongly agreed that the course will help them meet industry needs, that the professor helped achieve the course goals, and that they feel confident applying engineering design principles and processes to solve a new engineering problem. Grade Summary 54 Grade Summary Virtual Prototype & Presentation 40% Working Prototype & Presentation 40% 54 Virtual Prototype & Presentation 40% Working Prototype & Presentation 40% 54 Mechatronics and Applications: An International Journal (MECHATROJ), Vol. 2, No.1 Mechatronics and Applications: An International Journal (MECHATROJ), Vol. AUTHOR Kevin Craig attended the United States Military Academy at West Point, NY, earned varsity letters in football and baseball, and graduated with a B.S. degree and a commission as an officer in the U.S. Army. After serving in the military, he attended Columbia University and received the M.S., M.Phil., and Ph.D. degrees. While in graduate school, he worked in the mechanical-nuclear design department of Ebasco Services, Inc., a major engineering firm in NYC, and taught and received tenure at both the U.S. Merchant Marine Academy and Hofstra University. While at Hofstra, he worked as a research engineer at the U.S. Army Armament Research, Development, and Engineering Center (ARDEC) Automation and Robotics Laboratory. He received the 1987 ASEE New Engineering Educator Excellence Award, a national honor. In 1989, he joined the faculty at Rensselaer Polytechnic Institute (RPI). At RPI, he served as Director of Core Engineering, the first two years of the School of Engineering, and as Chair of the Engineering Science Interdisciplinary Department. As a tenured full professor of mechanical engineering, he taught and performed research in the areas of mechatronic system design and the modeling, analysis, and control of multidisciplinary engineering systems. With significant continuous funding from both industry and government, he developed the Mechatronics Program at RPI, which included an extensive teaching and research laboratory and several undergraduate and graduate courses in mechatronics. He collaborated extensively with the Xerox Mechanical Engineering Sciences Laboratory (MESL), an offshoot of Xerox PARC, during this time. During his 18 years at RPI, he graduated 37 M.S. students and 20 Ph.D. students. While at RPI, he authored over 30 refereed journal articles and over 50 refereed conference papers. Emphasis in all his teaching and research was on human-centered, model- based design, with a balance between theory and best industry practice. At RPI, he received the two highest awards conferred for teaching: the 2006 School of Engineering Education Excellence Award and the 2006 Trustees’ Outstanding Teacher Award. From 2007 to 2014, he wrote a monthly column on mechatronics for practicing engineers in Design News magazine. Over the past 30 years, he has conducted hands-on, integrated, customized, mechatronics workshops for practicing engineers nationally and internationally, e.g., at Xerox, Procter & Gamble, Rockwell Automation, Johnson Controls, Brady Corp., Pitney Bowes, and Siemens Health Care in the U.S., and at Fiat and Tetra Pak in Italy. [2] Craig, K., “Modeling and Simulation for Motor Selection,” Design News, June 2011. [2] Craig, K., “Modeling and Simulation for Motor Selection,” Design News, June 2011. [2] Craig, K., “Modeling and Simulation for Motor Selection,” Design News, June 2011 5. CONCLUSION 55 A new approach to mechanical engineering capstone design has been described and implemented. All seven four-person teams designed a four-bar mechanism for an assigned application. This allowed the instructor to teach fundamentals of kinematics and dynamics of 55 Mechatronics and Applications: An International Journal (MECHATROJ), Vol. 2, No.1 mechanisms, both forward and inverse, in particular, the four-bar mechanism. Nonlinear kinematic and dynamic analyses, both forward and inverse, were performed in Simulink. Control design was performed using MatLab, computer simulations were performed for the virtual prototype using Simulink, and the real-time control was programmed using LabVIEW with the NI myRIO controller. Students selected the brushed DC motors for their applications, developed the power electronics, interfaced the incremental optical encoder, and programed the real-time control code. A prerequisite for this course is the standard junior-level Machine Design course. In that course students learn about theories of failure, stress, strain, and deflection, as well as how to size a shaft and select bearings, couplings, and gears. They now put that into practice, as the mechanical design of the mechanism (links and bearings), support structure, and motor coupling was performed using these machine design principles. Performance requirements were continuously evaluated and updated. See mechanism pictures in the Appendix. Students had the opportunity for a hands-on, real-world mechatronic design experience, just as they will be expected to perform after graduation as real practicing engineers. REFERENCES [1] Craig, K., “Mechatronify Common Mechanisms,” Design News, August 2012. [1] Craig, K., “Mechatronify Common Mechanisms,” Design News, August 2012 APPENDIX Mechanical Engineering Senior Design Questionnaire 1- Strongly Agree 2- Agree 3-Neutral 4-Disagree 5-Strongly Disagree The purpose of this course is to develop in the student the attributes of a professional engineer in the application of their undergraduate engineering, mathematics, and science knowledge to the solution of a real-world engineering challenge using the model-based, integrated design approach, i.e., virtual prototype to working prototype, that is the hallmark of 21st-century engineering practice. _____ This course accomplished this goal. AUTHOR He is a Fellow of the ASME and a member of the IEEE and the ASEE. After a national search, in January 2008, he was chosen to be the Robert C. Greenheck Chair in Engineering Design, a $5 million endowed chair, at Marquette University. His mission was to integrate multidisciplinary design and discovery learning 56 Mechatronics and Applications: An International Journal (MECHATROJ), Vol. 2, No.1 throughout the entire college, in all years and in all departments. He transformed students, faculty, curricula, and facilities throughout the college and created a new engineering education mindset and culture for innovation. He was given the 2013 ASEE North-Midwest Best Teacher Award and the 2014 ASME Outstanding Design Educator Award, a society award. He graduated his 21st Ph.D.In the fall of 2014, he returned to the Hofstra University School of Engineering and Applied Science as a tenured full professor of mechanical engineering. He is the Director of the Mechatronics Laboratory, which he created with $1M funding from NYS / Hofstra, and also the Director of the Center for Innovation, a new center he created to collaborate with business and industry to foster innovation, where all intellectual property (IP) belongs to the sponsor. Recent clients include ThermoLift, P&G, and Toro. He has taught graduate courses as an Adjunct ME Professor at both Stony Brook University and NYU Tandon School of Engineering. Part A This course has provided me with: _____An ability to apply knowledge of mathematics, science, and engineering. _____An ability to apply knowledge of mathematics, science, and engineering. _____An ability to design and conduct experiments, as well as analyze and interpret data. _____An ability to design a system, component, or process to meet desired needs within realistic constraints such as economic, social, political, ethical, health and safety, manufacturability, and sustainability. _____ An ability to function on multidisciplinary teams. _____ An ability to identify, formulate, and solve engineering problems. _____ An understanding of professional and ethical responsibility. _____ An ability to communicate effectively. _____The broad education necessary to understand the impact of engineering solutions in a global, economic, environmental, and societal context. _____ A recognition of the need for, and an ability to engage in, life-long learning. _____ A knowledge of contemporary issues. _____ An ability to use the techniques, skills, and modern engineering tools necessary for engineering practice. _____An ability to design and conduct experiments, as well as analyze and interpret data. _____An ability to design a system, component, or process to meet desired needs within realistic constraints such as economic, social, political, ethical, health and safety, manufacturability, and sustainability. y _____ An ability to function on multidisciplinary teams. y _____ An ability to function on multidisciplinary teams. _____ An ability to identify, formulate, and solve engineering problems. _____ An understanding of professional and ethical responsibility. _____The broad education necessary to understand the impact of engineering solutions in global, economic, environmental, and societal context. _____ A recognition of the need for, and an ability to engage in, life-long learning. _____ A recognition of the need for, and an ability to engage in, life-long learning. A kno ledge of contemporar iss es _____ A knowledge of contemporary issues. _____ An ability to use the techniques, skills, and modern engineering tools necessary for engineering practice. Part B The following skills and knowledge, and their corresponding growths, are essential to a career as a practicing engineer. _____ Communication 57 Mechatronics and Applications: An International Journal (MECHATROJ), Vol. 2, No.1 _____ Teamwork _____ Project Management _____ Problem Solving _____ Organization _____ Leadership _____ Statics / Dynamics _____ Strength of Materials / Machine Design _____ Modeling, Analysis, and Control of Dynamic Systems _____ Electromechanics _____ Electronics _____ Fluid Mechanics _____ Thermodynamics _____ Heat Transfer _____ Computer Graphics (e.g., Solid Works) _____ MatLab / Simulink _____ LabVIEW _____ Real-Time Computer Programming Part C _____ This course will help you to meet industry needs. _____ This course resulted in improved student learning. _____ The professor in this course helped achieve the course goals. _____ You feel confident in applying engineering design principles an of a new engineering problem. _____ The approach used, i.e., to teach subject matter (e.g., mecha knowledge in the current design process, was very effective. _____ Teamwork _____ Project Management _____ Problem Solving _____ Organization _____ Leadership _____ Statics / Dynamics _____ Strength of Materials / Machine Design _____ Modeling, Analysis, and Control of Dynamic Systems _____ Electromechanics _____ Electronics _____ Fluid Mechanics _____ Thermodynamics _____ Heat Transfer _____ Computer Graphics (e.g., Solid Works) _____ MatLab / Simulink _____ LabVIEW _____ Real-Time Computer Programming Part C _____ This course will help you to meet industry needs. _____ This course resulted in improved student learning. _____ The professor in this course helped achieve the course goals. _____ You feel confident in applying engineering design principles and processes in the soluti of a new engineering problem. _____ The approach used, i.e., to teach subject matter (e.g., mechanisms) and then use th knowledge in the current design process, was very effective. 58 Mechatronics and Applications: An International Journal (MECHATROJ), Vol. 2, No.1 Mechatronics and Applications: An International Journal (MECHATROJ), Vol. 2, No.1 5 59 Mechatronics and Applications: An International Journal (MECHATROJ), Vol. 2, No.1 Mechatronics and Applications: An International Journal (MECHATROJ), Vol. 2, No.1 60
https://openalex.org/W1615678725	https://storage.googleapis.com/jnl-up-j-ijic-files/journals/1/articles/2180/submission/proof/2180-1-9082-1-10-20150521.pdf	English	null	Validation of the Primacy Care Resources and Support for Chronic Disease Self-Management (PCRS): An assessment tool for the quality of self-management support	International journal of integrated care	2,015	cc-by	770	Validation of the Primacy Care Resources and Support for Chronic Disease Self-Management (PCRS): An assessment tool for the quality of self- management support Maarten Voorhaar, Boehringer-ingelheim, CAPHRI School for Public Health and Primary Care, Maastricht University The Netherlands EWMA Bischoff, Radboud University Medical Centre, Department of Primary and Community Care, Nijmegen, The Netherlands A Visser, University College Roosevelt, Social Sciences, Middelburg, The Netherlands GM Asijee, Boehringer-ingelheim, CAPHRI School for Public Health and Primary Care, Maastricht University, The Netherlands GM Asijee, Boehringer-ingelheim, CAPHRI School for Public Health and Primary Care, Maastricht University, The Netherlands M Asijee, Boehringer-ingelheim, CAPHRI School for Public Health and Primary Care, Maastrich niversity, The Netherlands JWM Muris, CAPHRI School for Public Health and Primary Care, Maastricht University, The Netherlands JWM Muris, CAPHRI School for Public Health and Primary Care, Maastricht University, The Netherlands CP Van Schayck, CAPHRI School for Public Health and Primary Care, Maastricht University, The Netherlands CP Van Schayck, CAPHRI School for Public Health and Primary Care, Maastricht University, The Netherlands CP Van Schayck, CAPHRI School for Public Health and Primary Care, Maastricht University, The Netherlands Correspondence to: Maarten Voorhaar Boehringer-ingelheim, The Netherlands, E-mail: maarten.voorhaar@boehringer-ingelheim.com Correspondence to: Maarten Voorhaar Boehringer-ingelheim, The Netherlands, E-mail: maarten.voorhaar@boehringer-ingelheim.com Volume 15, 27 May 2015 Publisher: Uopen Journals URL: http://www.ijic.org Cite this as: Int J Integr Care 2015; Annual Conf Suppl; URN:NBN:NL:UI:10-1-117069 Copyright: Volume 15, 27 May 2015 Publisher: Uopen Journals URL: http://www.ijic.org Cite this as: Int J Integr Care 2015; Annual Conf Suppl; URN:NBN:NL:UI:10-1-117069 Copyright: Poster Abstract Abstract Introduction: The treatment of patients with a chronic condition is challenging the sustainability of healthcare systems worldwide. One possibility to utilize healthcare resources more efficiently is to enhance self-management of patients. However, self-management has not found a commonplace in chronic healthcare and one of the reasons is the lack of self-management support (SMS) given by healthcare professionals. To improve SMS, an assessment of the quality of current SMS in a healthcare organization is necessary. The Primary Care Resources and Support for Chronic Disease Self-management (PCRS) is an instrument to measure the quality of SMS, but evidence on its validity and reliability is limited. In this study we assessed the validity and reliability of the PCRS. Objective: The goal of this study is to assess the validity and reliability of the PCRS in measuring the quality of SMS by healthcare professionals. 15th International Conference on Integrated Care, Edinburgh, UK, March 25-27, 2015 1 International Journal of Integrated Care – Volume 15, 27 May – URN:NBN:NL:UI:10-1-117069 – http://www.ijic.org/ Method: In this observational study, we collected data on SMS in healthcare organization to investigate the face-, construct- and discriminant validity, as well as the reliability of the PCRS. We translated the PCRS from English to Dutch according to WHO recommendations. We asked health care professionals from general and specialized healthcare organizations to complete the PCRS, ACIC and CS-PAM and to assess the understandability and applicability of each individual item of the PCRS for face validity. The correlation between the self-management scale of the ACIC and the PCRS was a measure for construct validity, while the lack of a correlation between the CS- PAM and the PCRS was a measure for discriminant validity. To measure its reliability, we compared the correlation between the total score on t=0 to the total score on t=1, two weeks thereafter. Also, we assessed point reliability on t=0. Results: Data from 50 healthcare professionals showed that most items of the PCRS were considered to be completely understandable and relevant. The PCRS total score correlated strongly with the total score of the ACIC SMS subscale (r=.730, p<.001). We found no correlation between PCRS total and CS-PAM (r=.030, p>.005). Finally, Cronbach’s α and Guttmann’s λ, measuring respectively the test-retest and point-reliability, were high (r>0.9). Conclusion: We found evidence that the PCRS is a valid and reliable tool to assess the quality of SMS and is applicable for chronic healthcare. Abstract The PCRS consists of understandable items appropriate to assess SMS. It measures self-management support by healthcare professionals in an objective manner; it does not reflect the perception of healthcare professional on their self- management support. Discussion: Healthcare professionals who want to assess the quality of SMS, can use to the PCRS as a valid and reliable assessment tool. Based on its outcomes, the PCRS may direct the development of a tailored improvement plan, focusing on SMS during patient consultation as well as the organization of SMS. Future studies should investigate the added value of the PCRS in improving the quality of SMS. Keywords self-management; assessment; quality; questionnaire PowerPoint presentation http://integratedcarefoundation.org/resource/icic15-presentations 2 2 15th International Conference on Integrated Care, Edinburgh, UK, March 25-27, 2015
https://openalex.org/W4381713948	https://publications.pik-potsdam.de/pubman/item/item_28373_2/component/file_28405/28373oa.pdf	English	null	Can contract farming support sustainable intensification in agri-food value chains?	Ecological economics	2,023	cc-by	14,226	Chiara Sophia Weituschat a,b,, Stefano Pascucci b,c,d, Valentina Cristiana Materia b, Francesco Caracciolo e Chiara Sophia Weituschat a,b,, Stefano Pascucci b,c,d, Valentina Cristiana Materia b Francesco Caracciolo e a Department of Climate Resilience, Potsdam Institute for Climate Impact Research, Telegrafenberg A62, 14473 Potsdam, Germany b Business Management and Organisation Group, Wageningen University, Hollandseweg 1, 6706, KN, Wageningen, the Netherlands c Business School and Environment and Sustainability Institute, University of Exeter, ESI Building, Penryn, Campus Cornwall, TR10 9EZ, UK d Department of Management and International Business, University of Auckland Business School, Sir Owen G Glenn Building - Bldg 260, 12 Grafton Road, Auckland 1010, New Zealand , e Department of Agricultural Sciences, University of Naples Federico II, Via Universit`a 96 – 80055 Portici, Naples, Italy A R T I C L E I N F O SI generally refers to practices that maintain or increase yields, productivity and economic outputs, while minimising environ mental impacts and use of land (Pretty, 1997; Pretty and Bharucha, 2014; Tittonell, 2014). It broadly includes approaches and practices such as precision and climate-smart agriculture, integrated crop-pest management, and crop diversification, with a focus on adapting the use of agrochemicals and (bio-)technology to specific agro-ecological conditions (Garnett et al., 2013; Tittonell, 2014; Uphoff, 2014). The adoption of these practices often asks for incremental changes in prac tices by farmers, and thus may have the potential for a wider up-take, potentially affecting the agricultural system as a whole, and * Corresponding author at: Department of Climate Resilience, Potsdam Institute for Climate Impact Research, Telegrafenberg A62, 14473 Potsdam, Germany. E-mail address: sophia.weituschat@pik-potsdam.de (C.S. Weituschat). Ecological Economics 211 (2023) 107876 Ecological Economics 211 (2023) 107876 Available online 12 May 2023 0921-8009/© 2023 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). * Corresponding author at: Department of Climate Resilience, Potsdam Institute for Climate Impact Research, Telegrafenberg A62, 14473 Potsdam, Germany. E-mail address: sophia.weituschat@pik-potsdam.de (C.S. Weituschat). https://doi.org/10.1016/j.ecolecon.2023.107876 Received 10 February 2022; Received in revised form 17 April 2023; Accepted 1 May 2023 A R T I C L E I N F O Keywords: Contract farming Sustainable intensification Crop rotation Glyphosate Flower strips Discrete choice experiment Sustainable intensification aims to minimize the negative impacts of the current agricultural system while maintaining productivity and economic outputs. This study demonstrates that contract farming is a potential mechanism to support many, but not all, farmers in adopting sustainable intensification practices. A discrete choice experiment on a hypothetical value chain contract introducing three sustainable intensification practices, namely extended crop rotation, reducing agrochemicals and planting flower strips, was conducted with a sample of 314 north-Italian wheat farmers. The results show that permanently eliminating glyphosate from the plot under contract is strongly resisted by farmers, while farmers have less strong preferences between introducing legumes or oilseeds in rotation, and between temporary or permanent flower strips. Findings also indicate that farmers who are more educated, are not members of cooperatives and who generally prefer more flexible sales arrangements are unlikely to be triggered to adopt sustainable intensification practices through contract farming. Overall, this study indicates that while voluntary contract arrangements can be a potential tool to increase uptake of sustainable intensification practices, they will likely need to be complemented with more public policy intervention in order to bring sustainable intensification practices to scale. with higher economic vulnerability of farmers (de Roest et al., 2018). Sustainable intensification (SI) has become one of the leading ap proaches to transform these highly specialised and input-dependent agricultural systems (Garnett et al., 2013; Petersen and Snapp, 2015; Pretty, 1997). SI generally refers to practices that maintain or increase yields, productivity and economic outputs, while minimising environ mental impacts and use of land (Pretty, 1997; Pretty and Bharucha, 2014; Tittonell, 2014). It broadly includes approaches and practices such as precision and climate-smart agriculture, integrated crop-pest management, and crop diversification, with a focus on adapting the use of agrochemicals and (bio-)technology to specific agro-ecological conditions (Garnett et al., 2013; Tittonell, 2014; Uphoff, 2014). The adoption of these practices often asks for incremental changes in prac tices by farmers, and thus may have the potential for a wider up-take, potentially affecting the agricultural system as a whole, and with higher economic vulnerability of farmers (de Roest et al., 2018). Sustainable intensification (SI) has become one of the leading ap proaches to transform these highly specialised and input-dependent agricultural systems (Garnett et al., 2013; Petersen and Snapp, 2015; Pretty, 1997). Available online 12 May 2023 0921-8009/© 2023 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). https://doi.org/10.1016/j.ecolecon.2023.107876 Received 10 February 2022; Received in revised form 17 April 2023; Accepted 1 May 2023 1. Introduction In the last two decades, agricultural intensification and specializa tion have persisted, supported by market incentives and public policy interventions, and despite the mounting evidence of their negative im pacts on environment and climate (Kleijn et al., 2019; Rockstr¨om et al., 2017). Combined with a decline in soil health and increasing erosion, the threat to our food systems is growing (Davies, 2017). Among other factors, these impacts are due to reliance on monocultural systems and dependence on external inputs (Kleijn et al., 2019; Lanz et al., 2018), while economic profitability is often prioritized over social and ecological considerations (Struik and Kuyper, 2017; Lee et al., 2006). In European agricultural systems, increased agricultural intensification has also been observed in association with declining crop productivity and growing environmental costs (Antonini and Argil´es-Bosch, 2017), and C.S. Weituschat et al. Ecological Economics 211 (2023) 107876 agricultural value chains in particular (Duru et al., 2015; Pretty and Bharucha, 2014; Swinnen and Kuijpers, 2019). preferences. Doing so, we are the first to demonstrate the potential of contract farming as a mechanism encouraging the adoption of sustain able intensification in the European context, as well as providing com panies an indication for effective targeting for such contracts and policy makers for the limits of private sector initiatives. We have organised this paper as follows: We first give a brief overview of the concept of SI and then review relevant literature on the role of contract farming in the adoption of SI practices. Then, we present the context and methodology of this study, followed by the results of the choice experiment. Finally, we discuss the results and conclude. Despite its potential, so far, the adoption of SI practices is relatively low, as only 9% of global agricultural land is estimated to operate under some form of SI (Pretty et al., 2018), and uptake is especially low in countries generally characterised by industrialised agricultural systems. Despite their benefits, implementing such practices often implies (initial) increased costs for farmers (Rosa-Schleich et al., 2019), who struggle to reduce agrochemical use (Bakker et al., 2021; Ch`eze et al., 2020; Mann, 2018), diversify their crop rotations (Jouan et al., 2020; Lemken et al., 2017), or improve on-farm biodiversity (Kirchweger et al., 2020; Maz´e et al., 2021; Plaas et al., 2019). 2.1. Mapping sustainable intensification practices In this section, we will briefly discuss the concept of sustainable intensification (SI), as well as its related practices. SI has the overall aim to ensure food security for a growing population while minimising the negative environmental effects of industrial agriculture (Pretty, 1997; Rockstr¨om et al., 2017). It emphasizes the importance of adapting practices to local agroecological conditions when balancing production, productivity, and environmental costs and benefits (Pretty and Bhar ucha, 2014). However, due to this localised approach, many different practices have been discussed under the umbrella of SI (Tittonell, 2014; Wezel et al., 2015). This has led to confusion with regard to the actual meaning of the term and to what extent it actually differs from the status quo of industrialised agriculture (Petersen and Snapp, 2015). Some practices such as increasing the use of agrochemicals in previously low- input systems, which one might consider simply intensification, have also been described as SI (e.g. Mulwa et al., 2021; Struik and Kuyper, 2017). Its critics state that SI does not embody the paradigm shift necessary to mitigate and cope with the negative externalities of the current industrialised agricultural system (Levidow, 2015; Petersen and Snapp, 2015; Tittonell, 2014). However, while incremental, practices generally mentioned under this umbrella term, such as reduced and more precise use of agrochemicals, diversifying cropping systems, and improved seed varieties (Wezel et al., 2015), may still be a viable, or even necessary pathway towards more comprehensive and trans formational approaches, such as agroecology (Garibaldi et al., 2019). Despite the breadth of approaches classified as SI, several common characteristics of SI practices have been defined (Pretty and Bharucha, 2014): Extant literature, in fact, mostly discusses forms of value chain organisation, including contract farming (CF), that have enabled farmers to adopt new practices aiming to improve product quality, or comply with food safety standards (e.g. Kumar et al., 2018; Mulwa et al., 2021; Wossen et al., 2017). Only few studies address sustainable prac tices (see Banterle and Stranieri, 2013; Mazhar et al., 2021; Ricome et al., 2016 for singular examples). Further, only Ricome et al. (2016) and Banterle and Stranieri (2013) have focussed on the European context from a value chain perspective, while the majority of studies assessing (potential) adoption of sustainable practices in Europe usually consider government-led agri-environmental schemes (AES) (e.g. Bougherara et al., 2021; Kuhfuss and Subervie, 2018; Santos et al., 2016). 2.1. Mapping sustainable intensification practices Value chain and commercial private sector initiatives, such as CF, have found only limited space in the literature on adoption of sustain able practices, against the evidence that private actors play a significant role, e.g. by creating incentives, setting standards or exchanging knowledge (Banterle and Stranieri, 2013; Cholez et al., 2020). Against this background, this study aims to assess the potential of CF to support and incentivise the adoption of SI practices among farmers in Europe. Particularly, we draw on the case of a large-scale value chain- based initiative that has been implemented by a multi-national food manufacturer. More specifically, we have engaged with the Barilla Group and its recently launched sustainability-focussed Carta del Mulino initiative (CDM) (Barilla, 2021a). Through this programme, Barilla aims to reconfigure its value chain, including storage centres and millers, in order to incentivise farmers to adopt sustainable practices in line with an SI approach, focussing first on Europe (e.g. Italy, France and Germany). The design of a contract farming scheme has been key to Barilla’s strategy (Barilla, 2018a, 2018b; Pancino et al., 2019). Through continuous interaction with this case and its actors, starting in 2013, we have been able to engage with wheat producers located in northern Italy, the area designated for initial pilot studies and the launch of the CDM initiative. Based on an extensive survey of 314 north-Italian farmers, the design of which was informed by intense previous engagement with the case through interviews, focus groups and com pany documents, we have analysed farmers’ willingness to participate in a value chain-based contract farming scheme for SI. More specifically, this study pivots around three key questions: What is the potential of contract farming to incentivise the adoption of SI practices? Which practices can be effectively supported by contract farming? And which types of farmers are most likely to participate? 1. Introduction Additionally, a change in practices often requires the reorganisation of relations with down stream partners at value chain level, for example in the case of crop diversification and mixed cropping (Meynard et al., 2017), as well as the supply of suitable inputs from upstream, such as seeds and crop man agement systems (Magrini et al., 2016; Vanloqueren and Baret, 2009; Weituschat et al., 2022). Yet, while understanding the interplay between the adoption of SI practices and value chain (re-)organisation mecha nisms is key, it is, as of yet, still under-researched (Ricome et al., 2016; Swinnen and Kuijpers, 2019). 2.2. Contract farming and the adoption of SI practices 2.2. Contract farming and the adoption of SI practices This study analyses Barilla’s contract design for the Carta del Mulino initiative (CDM) aiming to make its soft wheat products more sustain able (Barilla, 2021a). CDM is in line with the concept of SI as it combines the aim to minimize environmental impact while maintaining or increasing economic value. This initiative followed several previous multi-stakeholder projects on value chain sustainability involving actors within and outside the company’s value chains (Barilla, 2018b, 2020; Pancino et al., 2019). Initiated by the marketing department, CDM was developed with input from an NGO, two universities and value chain partners. Based on the CDM initiative and extensive stakeholder engagement through focus group discussions with farmers, elevators and millers, and in continued consultation with researchers and key managers in Barilla, three SI practices were eventually selected for further consideration in this study, namely crop diversification, flower strips, and limits on the agrochemical glyphosate. In essence, the CDM initiative, presents a contract farming arrangement to farmers, based on ten rules specifically designed to increase wheat quality, crop diversity and protection for pollinators, and reduce agrochemical use (Barilla, 2021a, 2021b). In order to signal this to consumers, products from the CDM initiative are traced throughout the value chain, third-party certified and labelled accordingly. Rolled out in 2017, more than 1400 farmers currently deliver under the CDM contract (Barilla, 2021b). Despite the participatory approach used by Barilla to design the CDM contracts, and the initial success during the roll-out, the company also experienced some internal and external pressure to further explore op portunities to enhance CDM contracts. The key aspects for further analysis have been the ability of CDM to impose stricter measures and to attract new farmers to deliver under the CDM rules. Specifically, ques tions remained whether crop diversification is preferred with legumes or oil seeds, the temporary or permanent nature of flower strips and extent of reduction of the chemical glyphosate, among others. As part of the stakeholder engagement, our research team has designed the discrete choice experiment to analyse the role of the above-mentioned issues. By targeting this case and related farmers, we ensure that the hypothetical contract design tested here is perceived as realistic. Further, Alcon et al. 2.2. Contract farming and the adoption of SI practices (2020) show that consumers are indeed willing to pay a premium price, for example, for products from more diversified systems, indicating that this is also a potentially attractive option for the companies involved. There is increasing evidence on the relationship between of forms of value chain participation and the adoption of agricultural practices (Swinnen and Kuijpers, 2019). Often contractual arrangements are needed to provide premium prices to farmers in order to incentivise the adoption of practices and the implementation of (quality) standards (Banterle and Stranieri, 2013). Nonetheless, studies suggest that there is no blueprint contract to encourage adoption (Meynard et al., 2017; Swinnen and Kuijpers, 2019), and designing adequate contracts and value chain configurations to support the adoption of sustainable prac tices can be a complex process (Pancino et al., 2019). Empirically, several examples have started to illustrate the complex relationship, particularly between CF and farmers’ adoption of new practices (e.g. Bonjean, 2019; Mazhar et al., 2021; Mulwa et al., 2021). However, these studies largely focus on cases in emerging economies. Studies on the European context are still rather scarce and are mostly centred on the French context. Ricome et al. (2016) find a connection between marketing contracts and adoption of more sustainable prac tices. They argue that the risk-reduction of marketing contracts might allow for more risk-taking when it comes to French grain farmers’ adoption of low-input practices. Several studies analyse value chain organisation with regard to diversifying French cropping systems, such as Meynard et al. (2017) who analysed several case studies in France to show how diversifying cropping systems with minor crops requires a range of changes in the organisation of value chains. That analysis is complemented by Meynard et al. (2018) explaining how these required changes impede the uptake of such minor crops. Jouan et al. (2020) modelled how horizontal cooperation between crop and livestock farmers could diversify cropping systems by introducing legumes on French farms. Cholez et al. (2020) point to the importance of contract farming in the development and transfer of knowledge in legume pro duction when building new value chains, using a case study in Britany, France. These studies, while limited to the French context, illustrate that a change in practices at farm level often requires changes in the organistion of the value chain. 1 The unavailability of farmers’ contact data due to the Data Protection Act in force limits the possibility to use a more robust sampling design. Moreover, Italian farmers are quite unwilling to provide sensible business information and they generally agree to participate only if recommended by someone they trust. Therefore, we targeted farmers through their buyers and agronomic advisors. 2.1. Mapping sustainable intensification practices To answer these ques tions, we implemented a hypothetical choice experiment to assess three SI practices (crop diversification, flower strips, and limiting glyphosate use) as contract attributes, and relate them to farmer characteristics and attitudes as determinants of contract acceptance and attribute “(1) utilize crop varieties and livestock breeds with a high ratio of pro ductivity to use of externally and internally derived inputs; (2) avoid the unnecessary use of external inputs; (3) harness agroecological processes such as nutrient cycling, biological nitrogen fixation, allelopathy, predation and parasitism; i (4) minimize use of technologies or practices that have adverse impacts on the environment and human health; (5) make productive use of human capital in the form of knowledge and capacity to adapt and innovate and of social capital to resolve common landscape-scale or system-wide problems […]; and. (6) minimize the impacts of system management on externalities such as GHG emissions, clean water, carbon sequestration, biodiversity, and dispersal of pests, pathogens and weeds” (Pretty and Bharucha, 2014, p. 1577). Some of these practices are becoming increasingly relevant in the European context, with potential for broad uptake at scale. For example, diversifying crop rotations, particularly with legumes or oil crops, reduces pests and thus the use of pesticides (Pretty and Bharucha, 2014), in creases biodiversity (Garibaldi et al., 2019) and, when introducing le gumes, employs biological nitrogen fixation (Bedoussac et al., 2015; Lemken et al., 2017; Pretty and Bharucha, 2014). Planting flower strips increases on-farm biodiversity, reduces pests and creates habitats for pollinators (Gurr et al., 2016; Kirchweger et al., 2020). Finally, reducing use of agrochemicals, including glyphosate, can reduce their adverse Some of these practices are becoming increasingly relevant in the European context, with potential for broad uptake at scale. For example, diversifying crop rotations, particularly with legumes or oil crops, reduces pests and thus the use of pesticides (Pretty and Bharucha, 2014), in creases biodiversity (Garibaldi et al., 2019) and, when introducing le gumes, employs biological nitrogen fixation (Bedoussac et al., 2015; Lemken et al., 2017; Pretty and Bharucha, 2014). Planting flower strips increases on-farm biodiversity, reduces pests and creates habitats for pollinators (Gurr et al., 2016; Kirchweger et al., 2020). Finally, reducing use of agrochemicals, including glyphosate, can reduce their adverse 2 C.S. Weituschat et al. Ecological Economics 211 (2023) 107876 Ecological Economics 211 (2023) 107876 land size (Calvet et al., 2019; Kuhfuss and Subervie, 2018; Mack et al., 2020). 2.2. Contract farming and the adoption of SI practices Beyond France, Banterle and Stranieri (2013) discuss how Italian farmers’ adoption of integrated pest man agement practices for a private label also required a reconfiguration of contracts. Based on this literature, it is reasonable to suggest a rela tionship between value chain organisation and practice adoption, and there is still a lot of room to investigate the potential of contract farming to induce SI practice adoption, especially beyond the French context. 2.1. Mapping sustainable intensification practices effects on the environment and human health (Bakker et al., 2021; Ch`eze et al., 2020). Jointly, these practices thus reduce external inputs, make use of nitrogen fixation, reduce impacts on environment and human health, and improve biodiversity and reduce pests, all of which are SI principles (Pretty and Bharucha, 2014). 3.2. Research design, sampling and data collection As studies on contracts for sustainable practices with value chain partners are still rare, we also draw from studies on public contracts, such as AES, to inform our study design. It is well established in this literature that contract attributes influence farmers’ acceptance de cisions (e.g. Bougherara et al., 2021; Christensen et al., 2011; Santos et al., 2016). Beyond the contract itself, previous research has shown that participation in AES is also related to farmer and farm character istics (e.g. Kuhfuss and Subervie, 2018; Mack et al., 2020). For example, several studies have shown that farmers’ environmental attitudes in fluence their willingness to partake in AES (Calvet et al., 2019; Defrancesco et al., 2018; Was et al., 2021), as well as general perceptions of or resistance to contracts (Allen and Colson, 2019). This general resistance to contracts was also shown for marketing contracts, though unrelated to sustainable practices, for Italian cereal farmers, where resistance to the use of written contracts being due to an aversion to restrictions and low trust in contracts generally (Solazzo et al., 2020). Further, more standard control variables have also been linked to the participation in AES, such as gender, age, education, experience, and The research design and sampling has been strongly informed by the roll-out process of the CDM contracts, and the sample was thus drawn from farmers operating in the areas where the initiative was already in place. Given the purpose of our study, we did not aim for a regionally representative sample, but instead specifically targeted farmers engaged in industrial value chains, in order to explore the potential of contract farming among this specific group of farmers.1 Taking into account the sensitivity of issues discussed in the survey, including contract prefer ences and reservation prices, gaining access to farmers through a network known to them, was essential. We eventually sampled both from selected suppliers in the CDM value chain, as well as among 3 C.S. Weituschat et al. Ecological Economics 211 (2023) 107876 were carefully described to farmers in order to enable them to make an informed choice. Following standard procedure to calibrate the model ling approach, the level of the price premium was initially set based on stakeholder consultation, and then refined after the first set of surveys was collected (34 observations). Attributes of contracts and their levels are shown in Table 1. 3.4. Discrete choice model Once collected, farmers choices were empirically examined by adopting the theoretical framework of the Random Utility Model (McFadden, 2001) and the Conditional Logit Model for estimating model parameters (Train, 2003). Analytically, by showing a set of ‘A’ contract alternatives to the i-th farmers, the farmer’s utility associated with the alternative a can be represented as a linear function of all z attributes and levels characterizing contract a: The DCE in this study started with a concise description of a hypo thetical contract farming arrangement, framed as a contract with the farmers’ current buyer. As mentioned in the previous section, the analysis focused on three strategic aspects of the CDM contract, and namely crop rotation, flower strips and glyphosate restrictions, which Ui a = za ’Ω + ei a (1) (1) Ui a = za ’Ω + ei a where za is the vector of attributes characterizing the contract, Ω is a vector of unknown parameters, and ei a represents the stochastic error component. The model assumes that the i-th farmer chooses the contract alternative a rather than b since it maximizes their ‘expected utility’: Ui a ≥Ui b, where a and b alternatives ∈A and b ∕= a. Fig. 1. Location of data collection. Farmers’ choices are modelled in terms of probability: the proba bility that the i-th farmers prefers the contract a to b is due to the probability that the utility associated to the a alternative is higher (or equal) than the utility of the other proposed contract alternative: p(Ui a) = p{Ui a > max(Ui b, …,Ui A)}. In that way, the estimate of Ω provides the influence of the different contract attributes on the probability that the contract is chosen, allowing a measure of farmers’ preferences. i By considering preferences to be individual-specific, Ω parameters vector can be assumed as distributed in the sample according to a dis tribution function defined by a location (μ) and a scale (σ) parameter: Ui a = za ’Ωi + ei a (2) (2) Ui a = za ’Ωi + ei a with Ωi = Ω + νi. To allow correlation among the parameters, νi is assumed to be standard multivariate normally distributed νi ~ N(0, ΣΩ). 3.2. Research design, sampling and data collection comparable farmers in the same area, based on information provided by the company. This is due to the aim to reach both farmers currently targeted by CDM, as well as those in the same regions potentially tar geted in the future. Farmers were located in the northern regions of Italy, namely Emilia-Romagna, Lombardy, Piedmont and Veneto (see Fig. 1). Data was collected between December 2019 and February 2021. Due to the COVID-19 pandemic affecting northern Italy at the time, data collection was prolonged, and adjustments had to be made to the data collection strategy along the way. While data collection started face-to- face, we eventually had to adjust to data collection by phone. Appendix 1 presents details of the adjustments to the strategies that were needed to cope with the challenges related to the pandemic. The final sample contains 314 complete observations. The survey itself contained five sections which collected data on farmer and farm characteristics, the farmer’s value chain relationships, the discrete choice experiment, in formation on production and practices, and farmers’ attitudes, respectively. The DCE investigated farmers’ preferences by letting them choose between two contract schemes characterised by the four attributes with varying levels, plus an opt-out option, framed as the business-as-usual alternative. Levels of attributes change from one contract scheme to another following the Fedorov algorithm (Carlsson and Martinsson, 2003; Cook and Nachtsheim, 1980) that maximizes the D-efficiency of the design according to the covariance matrix of the conditional logit model. The resulting design identified 24 choice tasks organized in six blocks. Farmers were randomly assigned to one of the six blocks, each one containing four choice tasks. Fig. 2 shows an example of a choice set. Based on the presented choice set, respondents were asked to choose the preferred contract alternative or the opt-out option. This process was repeated for each choice task by each respondent, thus delivering (314 × 4) 1256 overall stated choices from 314 farmers. 3.3. Discrete choice experiment The questionnaire presented to farmers in the sample included a discrete choice experiment (DCE) to understand the potential of CF to incentivise the adoption of SI practices, analysing farmers’ willingness to comply with specific restrictions. While initially developed by mathematicians in the 1960s (Luce and Tukey, 1964), DCEs have largely been used by economists to measure preferences and willingness to exchange attributes for a diverse range of products and services. Some examples of DCEs include, among others, health applications (Green and Gerard, 2009), job and food choices (Jaung et al., 2019; Miranda et al., 2012), and contract design (van den Broeck et al., 2017). MVAs i = xi’βs + us ii More specifically, based on the literature presented in Section 2.2, a range of farmer and farm characteristics were used to populate the x matrix: farmer’s gender, age, experience working in agriculture (in years) to proxy a farmer’s knowledge and skills, and the farmer’s formal education, with the latter coded into three categories: No formal degree, at least a high school degree, and a university degree. The regressions also include among the covariates: whether the farmer was working on the farm full time (equivalent to a 5 day work week) to control for the time spent on the farming business, whether they were a member of a farmer cooperative or association since this influences the farmers’ current value chain, whether they were already involved in CDM value chain and whether the farm held any certification (e.g. GlobalGAP, organic, or similar) to proxy the farmers’ familiarity with the adminis trative burden of such a CF scheme. Moreover, three attitudinal scales on farmers’ environmental attitudes, preferences for transaction conditions around the sale of wheat, and satisfaction with the current sales arrangement for wheat have been included. These attitudinal scales are each based on a set of statements and 5-point Likert scales asking to what extent the farmer agrees with each statement. Standardised means were used to combine the items into single indicators for each attitudinal scale. 3.4. Discrete choice model Individual model parameters Ωi were thus estimated using the maximum likelihood estimator assuming conditional logit random parameter specifications (Train, 2003), while the opt-out option was empirically modelled by introducing an alternative-specific constant (ASC) into the utility function for the opt-out alternative (Campbell and Erdem, 2019). From the estimated parameters, for each farmer, the marginal value of the attribute in monetary terms (MVA) is calculated. It measures the trade-off between the presence of the s-th contract attribute and the base price. Analytically, it can be calculated taking the ratio parameter esti mated for the non-monetary contract attributes to the price parameter multiplied by minus one, as follows: MVAs i = −Ωs i/ Ωprice (3) Fig. 1. Location of data collection. 4 Ecological Economics 211 (2023) 107876 Table 1 Overview of contract attributes. Attribute Level A Level B Price premium Market price + 10€ per ton Market price + 30€ per ton Crop rotation At least one legume included in 5 year rotation. At least one oil crop included in 5 year rotation. Flower strips Permanent flower strips of at least 3% of contracted soft wheat area located inside the farm. Temporary flower strips of at least 3% of contracted soft wheat area located inside or next to the wheat field. Ban on glyphosate Temporary ban of glyphosate in the parcel under contract from 60 days prior to sowing until after harvest. Permanent ban of glyphosate in the parcel under contract. C.S. Weituschat et al. Ecological Economics 211 (2023) 107876 C.S. Weituschat et al. C.S. Weituschat et al. Table 1 Overview of contract attributes. Attribute Level A Level B Price premium Market price + 10€ per ton Market price + 30€ per ton Crop rotation At least one legume included in 5 year rotation. At least one oil crop included in 5 year rotation. Flower strips Permanent flower strips of at least 3% of contracted soft wheat area located inside the farm. Temporary flower strips of at least 3% of contracted soft wheat area located inside or next to the wheat field. Ban on glyphosate Temporary ban of glyphosate in the parcel under contract from 60 days prior to sowing until after harvest. Permanent ban of glyphosate in the parcel under contract. Table 1 Overview of contract attributes. Overview of contract attributes. Attribute Level A Price premium Market price + 10€ per ton Crop rotation At least one legume included in 5 year rotation. Flower strips Permanent flower strips of at least 3% of contracted soft wheat area located inside the farm. Ban on glyphosate Temporary ban of glyphosate in the parcel under contract from 60 days prior to sowing until after harvest. Market price + 30€ per ton At least one oil crop included in 5 year rotation. Temporary flower strips of at least 3% of contracted soft wheat area located inside or next to the wheat field. Permanent ban of glyphosate in the parcel under contract. Fig. 2. Example of choice set presented to farmers in Qualtrics. Fig. 2. Example of choice set presented to farmers in Qualtrics. Fig. 2. Example of choice set presented to farmers in Qualtrics. Similarly, the same procedure has been followed to calculate the marginal value of the opt-out alternative. While recognizing that several aspects may influence respondents choosing to opt out, such as the perceived complexity of the task, strategic or protest responses, we believe this analysis contributes to a better understanding of farmers willingness to prefer the status quo, rather than the sustainability- focussed CF scheme. With MVAs i < 0, it indicates the maximum amount of the base price per ton that the farmer is willing to give up in order to get that attribute in the contract, while with MVAs i > 0, it in dicates the extra amount on the base price that farmers require to accept that attribute in the contract. Further insights can be gained via in spection of the empirical cumulative distribution function (eCDF) of the MVAs associating each euro added to the base price with the share of farmers that are willing to accept that attribute in the contract. better identification of drivers of heterogeneity in preferences. i In detail, following Curtis et al. (2020), a system of regression equations was set up, where the marginal values assigned to each farmer’s MVAs i for each non-monetary contract attribute were analysed against farmer and farm characteristics using the seemingly unrelated estimator: MVAs i = xi’βs + us i (4) (4) MVAs i = xi’βs + us ii 4.1. Results of the discrete choice model This section presents the results of the discrete choice model. Esti mates from the random parameter conditional logit analysis show that farmers strongly prefer a temporary restriction on glyphosate use over permanent ones. In fact, on average, to impose a permanent ban on glyphosate covering the entire rotation on the plot under contract would require a price premium of 33€/ton, with 19€/ton necessary to enrol just the most willing 25% of farmers. Further, temporary flower strips are preferred over permanent ones. This preference is less strong, however, if compared to the glyphosate ban. Only approximately 5€/ton would be needed, on average, to switch farmers from temporary to permanent flower strips. Finally, legume production in rotation is preferred over oil seed production. As can be seen in the empirical cumulative density function (see Fig. 5), less than 20% of farmers request any price pre mium at all to introduce legumes. Finally, the opt-out alternative is less preferred than entering the contract, meaning that overall farmers, on average, see value in contracting in order to implement SI practices for a For flower strips and the opt-out option, we again find similar de terminants for both. Coefficients are higher for the opt-out alternative which are generally strongest across the results of all regressions. Here we find those with higher education more likely to oppose permanent flower strips and less likely to want to enter the contract at all, preferring the opt-out alternative. Farmers that are members in a cooperative or farmers association are less likely to oppose permanent flower strips. They also have a strong preference to enter one of the proposed con tracts. This is also reflected in farmers’ transaction preferences: those preferring more security in their sales arrangements for wheat are also more likely to want to enter the contracting arrangement, and are less likely to resist permanent flower strips. Similarly to the results observed for use of glyphosate and oilseeds in rotation, farmers’ environmental attitudes are related to preferences for flower strips. Farmers’ belief to be able to affect environmental outcomes and willingness to organise to achieve slightly reduces resistance to permanent flower strips. These attitudes also strongly relate to farmers’ preference to enter the contract over the opt-out alternative. a Source: Farm Accountancy Data Network, 2020. 4.2. Heterogeneity of farmers preferences To examine the determinants of the marginal values of contract at tributes and the opt-out option, we used seemingly unrelated regression estimates (Table 6). Results highlight similar statistically significant determinants for permanent glyphosate bans and introduction of oil seeds over legumes, with coefficients higher for glyphosate, indicating farmers have slightly stronger preferences regarding glyphosate use. All else equal, male farmers are more resistant to both stronger glyphosate restrictions and oil seed introduction in rotation instead of legumes. We find the same effect for full time farmers. Certification holders are less likely to oppose a permanent ban on glyphosate and less likely to prefer legumes. Farmers who believe to have effects on environmental out comes and are willing to engage with others to improve the environ ment, and those more satisfied with their current sales arrangement are less resistant to permanent bans on glyphosate and oil seeds in rotation. Being interviewed by phone rather than face-to-face is also related to a higher resistance to a permanent ban on glyphosate as well as oil seeds in rotation. Finally, the only difference we see in determinants for these two regressions is with regard to whether farmers are currently participating in the CDM programme, which is not significantly related to preferences for glyphosate use but significantly reduces resistance to oil seeds in rotation. li Table 3 Table 3 Descriptive statistics of categorical variables (n = 314). price premium. Based on this analysis, over 60% of farmers in the sample see an added value in the presented contracts (Fig. 7). However, Fig. 7 also shows that of those farmers who demand a price premium, the majority requires one above 50€ per ton. Tables 4 and 5 show the esti mates for this analysis. Fig. 4-7 show the empirical cumulative distri bution functions for each contract attribute and the opt-out alternative. price premium. Based on this analysis, over 60% of farmers in the sample see an added value in the presented contracts (Fig. 7). However, Fig. 7 also shows that of those farmers who demand a price premium, the majority requires one above 50€ per ton. Tables 4 and 5 show the esti mates for this analysis. Fig. 4-7 show the empirical cumulative distri bution functions for each contract attribute and the opt-out alternative. Finally, a dummy variable was included in the model to control for any systematic differences on MVAs due the adjustment of the data collection strategy that obliged us to use phone interviews rather than face-to-face interviews. Descriptive statistics for all variables used can be found in Table 2 and Table 3. Where available, we also mention data from the Farm Accountancy Data Network (2020) representative for the same regions for comparison. Fig. 3 presents the proposed conceptual relationships between variables as indicated in this chapter. 4.2. Heterogeneity of farmers preferences 3.5. Analysis of farmers’ preference heterogeneity In order to deepen the understanding of the heterogeneity of CF attribute preferences among farmers, a set of regressions was applied. This two-steps approach, using separate regression models to analyse preference heterogeneity using a random parameter specification, was suggested by Campbell (2007) and is often adopted in the literature (Czajkowski et al., 2017; Jiang and Chen, 2016; Yao et al., 2014). Campbell (2007) has shown how the use of the two-stage approach adds considerable explanatory power over standard methods that directly include individual-specific variables into the conditional logit. Mean while, Curtis et al. (2020) have highlighted the advantage of the two- stage approach being substantially less computationally demanding. The same authors mentioned that the two-stage approach allows for a 5 Ecological Economics 211 (2023) 107876 b Mean and standard deviation of farm size are strongly affected by two outlier observations with large land holdings. Without these observations the mean drops to 55.65 ha with a standard deviation of 82.90 which is relatively close to the official statistics provided by FADN. The further analysis is not affected by these observations. C.S. Weituschat et al. For environmental attitudes, the set of questions is inspired by the idea that environmental action is affected by farmers’ beliefs as to whether their own individual actions and production can have an impact on improving the environment, and whether environmental ac tion is better done collectively (Cleveland et al., 2020; Despotovi´c et al., 2021; Poteete and Ostrom, 2004). The attitudinal scale for transaction conditions measured the farmer’s preferences for uncertainty and asymmetric information in sales transactions, focussing on price security and comparisons, transparent quality control, and plannability of sales activities (Cembalo et al., 2014; Pascucci et al., 2016). Lastly, the scale for satisfaction with the current buyer measures whether the sales prices are perceived as fair and at least cover costs, whether quality re quirements from the current buyer are clear and whether the farmer believes this to be a long-term sales relationship. This variable was included in order to estimate the role of the current relationship with the buyer in the farmer’s decision-making, since the DCE was framed as a contract with this current buyer, and the farmer’s satisfaction with this relationship is likely to influence whether the farmer would like to intensify this arrangement. The individual items forming the indicators can be found in appendix 1. Table 3 Descriptive statistics of categorical variables (n = 314). Variable Categories Gender Male Female 95.54% 4.46% (Official statisticsa) (84.66%) (15.34%) Education No degree High school degree University degree 23.25% 70.06% 6.69% Yes No Full time farmer 86.62% 13.38% Cooperative membership 71.97% 28.03% Presence of Certificationb 36.94% 45.54% CDM farm 32.48% 67.52% Phone interview 35.99% 64.01% a Source: Farm Accountancy Data Network, 2020. b average given to missing values. 4.1. Results of the discrete choice model Currently participating in the CDM pro gramme significantly reduces resistance to permanent flower strips, as well as, expectedly, significantly increasing farmers’ preference to Table 2 Descriptive statistics of continuous variables (n = 314). Variable Mean Std. Dev. Min Max Age (years) 51.24 12.97 19 84 Experience in agriculture (years) 28.78 13.43 1 75 Farm size, UAA (ha)b 77.89 286.67 2.5 3500 (Official statisticsa) (50.90) (68.92) Collective environmental attitudes 0 0.61 −1.75 1.55 Transaction preferences 0 0.73 −1.85 1.35 Satisfaction with current buyer 0 0.73 −1.57 1.46 Table 2 Descriptive statistics of continuous variables (n = 314). Table 2 Table 2 Descriptive statistics of continuous variables (n = 314). 6 C.S. Weituschat et al. Ecological Economics 211 (2023) 107876 Ecological Economics 2 Fig. 3. Conceptual relationships between variables. Fig. 3. Conceptual relationships between variables. participate in the hypothetical contract over the opt-out option. Finally, being interviewed by phone instead of face-to-face decreases resistance to permanent flower strips, and increases farmers’ preference for participation in the contract over the opt-out option. Table 4 Random parameter conditional logit estimates. Attribute Coef. Ω std.err p-value Price Premium 0.053 0.009 <0.001 Use of Glyphosate (Permanent) −1.741 a 0.236 <0.001 Flower strips (Permanent) −0.276 a 0.155 0.075 Crop Rotation (Oilseeds) −0.29 a 0.167 0.083 Opt-out (business as usual) −2.951 a 0.708 <0.001 σ(Glyphosate) 1.870 0.257 <0.001 σ(Flower strip) 0.269 0.174 0.123 σ(Rotation) 0.323 0.209 0.123 σ(Opt-out) 7.999 1.041 <0.001 a : computed as mean value (μ) of the parameter distribution. Table 5 Marginal value of the attribute (€/tons). Attribute Mean std. err 95% conf interval I quantile III quantile Use of Glyphosate (Permanent) 33.18 1.22 30.78 35.59 19.04 53.41 Flower strips (Permanent) 5.33 0.20 4.93 5.72 1.74 9.46 Crop Rotation (Oilseeds) 5.57 0.22 5.14 6.00 2.98 8.24 Opt-out 54.30 7.07 40.39 68.21 −94.07 176.44 Fig. 4. Empirical cumulative distribution function of the marginal value of using glyphosate (95% confidence interval). Table 4 Random parameter conditional logit estimates. Attribute Coef. Ω std.err p-value Price Premium 0.053 0.009 <0.001 Use of Glyphosate (Permanent) −1.741 a 0.236 <0.001 Flower strips (Permanent) −0.276 a 0.155 0.075 Crop Rotation (Oilseeds) −0.29 a 0.167 0.083 Opt-out (business as usual) −2.951 a 0.708 <0.001 σ(Glyphosate) 1.870 0.257 <0.001 σ(Flower strip) 0.269 0.174 0.123 σ(Rotation) 0.323 0.209 0.123 σ(Opt-out) 7.999 1.041 <0.001 a : computed as mean value (μ) of the parameter distribution. participate in the hypothetical contract over the opt-out option. Finally, being interviewed by phone instead of face-to-face decreases resistance to permanent flower strips, and increases farmers’ preference for participation in the contract over the opt-out option. Table 4 Random parameter conditional logit estimates. Attribute Coef. Table 4 Random parameter conditional logit estimates. Table 5 Marginal value of the attribute (€/tons). Attribute Mean std. err 95% conf interval I quantile III quantile Use of Glyphosate (Permanent) 33.18 1.22 30.78 35.59 19.04 53.41 Flower strips (Permanent) 5.33 0.20 4.93 5.72 1.74 9.46 Crop Rotation (Oilseeds) 5.57 0.22 5.14 6.00 2.98 8.24 Opt-out 54.30 7.07 40.39 68.21 −94.07 176.44 Table 5 Marginal value of the attribute (€/tons). Table 2 Ω std.err p-value Price Premium 0.053 0.009 <0.001 Use of Glyphosate (Permanent) −1.741 a 0.236 <0.001 Flower strips (Permanent) −0.276 a 0.155 0.075 Crop Rotation (Oilseeds) −0.29 a 0.167 0.083 Opt-out (business as usual) −2.951 a 0.708 <0.001 σ(Glyphosate) 1.870 0.257 <0.001 σ(Flower strip) 0.269 0.174 0.123 σ(Rotation) 0.323 0.209 0.123 σ(Opt-out) 7.999 1.041 <0.001 a : computed as mean value (μ) of the parameter distribution. Table 5 Marginal value of the attribute (€/tons). Attribute Mean std. err 95% conf interval I quantile III quantile Use of Glyphosate (Permanent) 33.18 1.22 30.78 35.59 19.04 53.41 Flower strips (Permanent) 5.33 0.20 4.93 5.72 1.74 9.46 Crop Rotation (Oilseeds) 5.57 0.22 5.14 6.00 2.98 8.24 Opt-out 54.30 7.07 40.39 68.21 −94.07 176.44 Fig. 4. Empirical cumulative distribution function of the marginal value of using glyphosate (95% confidence interval). Fig. 4. Empirical cumulative distribution function of the marginal value of using glyphosate (95% confidence interval). Fig. 4. Empirical cumulative distribution function of the marginal value of using glyphosate (95% confidence interval). Table 4 Random parameter conditional logit estimates. Attribute Coef. Ω std.err p-value Price Premium 0.053 0.009 <0.001 Use of Glyphosate (Permanent) −1.741 a 0.236 <0.001 Flower strips (Permanent) −0.276 a 0.155 0.075 Crop Rotation (Oilseeds) −0.29 a 0.167 0.083 Opt-out (business as usual) −2.951 a 0.708 <0.001 σ(Glyphosate) 1.870 0.257 <0.001 σ(Flower strip) 0.269 0.174 0.123 σ(Rotation) 0.323 0.209 0.123 σ(Opt-out) 7.999 1.041 <0.001 a : computed as mean value (μ) of the parameter distribution. 5. Discussion Our research was designed pivoting around three key questions, to (1) analyse the potential of contract farming to incentivise the adoption of SI practices; (2) identify which practices can be effectively supported by contract farming; and (3) to identify the types of farmers most likely to participate in a sustainability-focussed contract farming scheme, such as the CDM initiative. Our results indicate that contract farming indeed has potential to support and incentivise the adoption of SI practices. However, the evidence gathered suggests that some practices seem to be challenging farmers’ adoption more than others, while highlighting the relevance of designing contract farming arrangements taking into ac count farmers’ characteristics. We start by discussing the former and then we move into a commentary on the latter, concluding the section by reconnecting our discussion to key contributions to the extant literature. Looking at the practices tested in this study, glyphosate limitations seem to be the most difficult for farmers to accept. This indicates that glyphosate is a central element in the cropping system of farmers in this sample which they are rather unwilling to give up. This result is in line with other studies on the adoption of SI practices related to use of ag richemicals (Bakker et al., 2021; Ch`eze et al., 2020; Mann, 2018), and indicate where a key challenge in sustainability transitions may lay. There may also be an interaction between the adoption of flower strips, which may be a source of weeds, and the ban on glyphosate, as the adoption of the former may create a reluctance to accept the latter. While the literature on SI practices suggests an incremental approach to the adoption process, some of these practices for conventional farmers are more disruptive than others. Banning agrochemicals or severe lim itations to their use, like in the case of the glyphosate in our study, is indeed perceived as a strong, perhaps unfeasible, constraint to some farmers’ operations. Agrochemical management is likely to be a key aspect to consider in further articulation of a contract farming approach to SI practice adoption. Farmers’ preference to include legumes in their rotation is encouraging, given the more extensive environmental bene fits of legumes. According to some stakeholders’ assessment, this may also be due to the regional circumstances of northern Italy, where agro- ecological conditions are fairly suitable for legumes and where there is cultural recognition of legume production. Table 5 Fig. 4. Empirical cumulative distribution function of the marginal value of using glyphosate (95% confidence interval). participate in the hypothetical contract over the opt-out option. Finally, being interviewed by phone instead of face-to-face decreases resistance to permanent flower strips, and increases farmers’ preference for participation in the contract over the opt-out option. Fig. 5. Empirical cumulative distribution function of the marginal value of crop rotation (95% confidence interval). C.S. Weituschat et al. Ecological Economics 211 (2023) 107876 C.S. Weituschat et al. C.S. Weituschat et al. Fig. 5. Empirical cumulative distribution function of the marginal value of crop rotation (95% confidence interval) C.S. Weituschat et al. 5. Discussion This may also be due to an increasing recognition of the overall economic benefits of legumes in rotations as indicated by, e.g., Cortignani and Dono, 2020 and Zander et al., 2016. Further, despite being low in recent decades, demand for legumes in food products in Europe is slowly growing under shifts to “healthier” and “more sustainable” diets (Lascialfari et al., 2019; Fer reira et al., 2021; Cusworth et al., 2021). Fig. 5. Empirical cumulative distribution function of the marginal value of crop rotation (95% confidence interval). Fig. 6. Empirical cumulative distribution function of the marginal value of flower strips (95% confidence interval). Fig. 6. Empirical cumulative distribution function of the marginal value of flower strips (95% confidence interval). Fig. 7. Empirical cumulative distribution function of the marginal value of the opt-out option (95% confidence interval). When it comes to farm and farmer characteristics, gender, education and being a fulltime farmer played a role in farmers preferences, which has also been found in previous studies (Calvet et al., 2019; Kuhfuss and Subervie, 2018; Mack et al., 2020). While more highly educated farmers have at times been found to be more open to participating in environ mental schemes (e.g. Calvet et al., 2019), in this sample we find them to be more likely to resist contracting. Potentially, they may experience higher opportunity cost due to having more options at their disposal. Full-time farmers may be more resistant to stricter measures since their entire professional life is organised around their cropping system, thus making any changes to that system more profound. Certified farmers are possibly less resistant to stricter measures since they may already be used to adjusting to bigger changes in their cropping system, if they have already done so in the past to gain certification. Alternatively, in the case of organic, they may already go without agrochemicals entirely, and a complete removal of glyphosate from the given plot may thus not require any change at all. Fig. 7. Empirical cumulative distribution function of the marginal value of the opt-out option (95% confidence interval). Despite often theorised and found to impact adoption of innovative contracts and practices, the farmer’s age and agricultural experience played no role in our model (e.g. Calvet et al., 2019; Mack et al., 2020; Murphy et al., 2011). Also, farm size had no effect on preferences for the contract or individual preferences (in contrast to e.g. 5. Discussion Kuhfuss and 8 Ecological Economics 211 (2023) 107876 C.S. Weituschat et al. C.S. Weituschat et al. Table 6 Determinants of marginal value of the attribute in monetary terms (MVA), Seemingly unrelated regression estimates. Glyphosate (permanent) Flower strips (permanent) Rotation (oilseeds) Opt-out (Business as usual) Coef Std.err p-value Coef Std.err p-value Coef std.err p-value Coef Std.err p-value Age 0.111 0.155 0.476 0.016 0.026 0.536 0.027 0.027 0.315 −0.432 0.930 0.643 Gender (male) 10.525 5.817 0.07 1.251 0.994 0.208 2.444 1.023 0.017 −41.800 34.908 0.231 Education −2.272 2.634 0.388 1.125 0.450 0.012 0.119 0.463 0.798 ¡38.672 15.806 0.014 Full time 8.854 3.564 0.013 0.578 0.609 0.342 1.815 0.627 0.004 −21.833 21.390 0.307 Cooperative membership −0.139 2.788 0.96 ¡1.189 0.476 0.012 −0.584 0.490 0.234 38.879 16.730 0.020 Experience in agr. −0.114 0.150 0.448 −0.005 0.026 0.85 −0.022 0.026 0.396 0.119 0.902 0.895 Farm size 0.003 0.004 0.464 0.001 0.001 0.229 0.001 0.001 0.206 −0.028 0.024 0.251 Presence of Certification 0.625 2.993 0.835 −0.787 0.511 0.124 −0.249 0.526 0.636 29.057 17.961 0.106 Collective environmental attitudes ¡4.057 2.109 0.054 ¡0.657 0.360337 0.068 ¡1.014 0.371 0.006 22.168 12.659 0.080 Transaction preferences 1.390 1.778 0.434 ¡0.557 0.303647 0.066 −0.005 0.313 0.988 23.068 10.668 0.031 Satisfaction with current buyer ¡2.153 1.122 0.056 −0.224 0.301255 0.457 ¡0.483 0.276 0.080 9.756 10.584 0.357 CDM farm −2.929 2.920 0.316 ¡1.120 0.499 0.025 ¡1.038 0.513 0.043 33.338 17.522 0.057 Phone interview 14.684 2.901 0.001 ¡2.484 0.496 0.001 1.445 0.510 0.005 87.588 17.410 0.000 Cons. 10.200 9.326 0.274 4.405 1.593 0.006 1.089 1.640 0.507 82.360 55.968 0.141 R2: 0.237(Glyphosate), 0.169(Flower strips), 0.245(Rotation), 0.17 (Opt-out) #obs 314 Note: Statistically significant coefficients (p < 0.10) are reported in bold. overall prefer more autonomy and flexibility are less inclined to engage in more coordinated value chain mechanisms, whether that is a coop erative or contract farming for SI. Subervie, 2018; Mack et al., 2020; Murphy et al., 2011). We also confirm the role of environmental attitudes found by e.g. Defrancesco et al. (2018), Was et al. (2021) and Despotovi´c et al. (2021). Effects we found for cooperative members and transaction preferences are in line with previous studies in which general attitudes towards contracts shaped farmers’ preferences for specific contract offers (Allen and Colson, 2019; Solazzo et al., 2020). Finally, we found that satisfaction with the current buyer relationship makes farmers more likely to accept stricter contract conditions which echoes Calvet et al. 5. Discussion (2019), who emphasise the importance of trust in the contracting institutions: trust seems more important the more profound the changes requested from the farmer. As one would expect, already participating in the CDM contract reduces resistance to some measures (permanent flower strips and oil seeds in rotation) and is related to being more willing to participate also in this hypothetical contract. Interestingly, it is not significantly related to farmers’ preference regarding temporary or permanent glyphosate ban. This may be since farmers may have reservations on restricting glyph osate at all, rather than whether that restriction is temporary or per manent. This effect would only be reflected in farmers’ general willingness to participate in the contract. Our results indicate that a price premium will indeed be necessary to induce farmers’ acceptance of contract farming for SI. While Alcon et al. (2019, 2020) estimate that consumers are willing to pay more for products from more sustainable and diversified production, also in Italy, it remains to be seen whether the premium paid by consumers can fully compensate the price increase needed for farmers. This may even be exacerbated given that likely not the entire price increase paid by con sumers will reach farmers. Any premium paid by consumers will likely also need to compensate increased coordination costs along the value chain, as well as being subject to effects of imbalance in market power and negotiations among value chain actors. g g Yet, looking at our results, we can conclude that indeed a contract farming approach to facilitate adoption of SI practices is not necessarily an option for all farmers, and surely is highly dependent on the type of SI practice under consideration. Almost 40% of farmers in the sample require a likely prohibitive price premium and can thus be considered to be unwilling to engage in contracting for SI. These are mostly farmers that, all else equal, have more formal education, are not members of cooperatives and generally prefer more flexibility in their sales ar rangements. This echoes results from Solazzo et al. (2020) who found that Italian wheat farmers in their sample prefer the autonomy granted by foregoing written contracts while at the same time still building on trust-based long-term relationships with their buyer. 2 Other answers were that the contract was too risky (7%), the administrative burden too high (5%), preferring direct sales and/or on farm processing (4%), and preferring to sell under the organic label (4%). Multiple answers were possible and the question was only asked to farmers who rejected both contract options in all four choice tasks. 85 farmers answered this question. 5. Discussion This seems to imply that a significant share of farmers is unlikely to be attracted by contracts that require written documentation, e.g., in order to provide certification of the practices used in crop production, which may limit the effect contract farming can have on practice adoption. It is still un clear, however, whether the trust in long-term buyers itself, rather than a contract, may be an avenue to reach these farmers and whether that will suffice to induce a change in practices, considering that some farmers may also be principally opposed to certain (agro-ecological) practices (Jaeck and Lifran, 2014). Thus, to maximise uptake of sus tainable practices, heterogenous preferences for both contracts and practices need to be taken into account (Mack et al., 2020; de Salvo et al., 2018). The similarity of determinants for glyphosate restrictions and crop rotations could be explained by the fact that both these practices are rather disruptive to farm management. Whether flower strips are tem porary or permanent, their implementation could be considered less disruptive, and its determinants may therefore be more connected to whether the farmer is willing to engage in any kind of contracting that restricts choices in farm management. If one is opposed to those con tracts in general, one may also be more sensitive to further restrictions, even if they are rather minor. The negative association between wanting to participate in the contract and preferring more flexibility in trans actions is further echoed by the fact that, when asked why none of the contracts in the four choice tasks were selected, the most common answer from farmers was that they preferred to maintain more flexibility in their sales arrangement (59% of farmers). The individual practices were also mentioned as reasons to not want to enter the contract at all. Here reasons related to the rotation requirement were most common (42%), followed by glyphosate restrictions (34%) and flower strips (27%).2 The premium being too low was only mentioned by 12% of farmers. These results support the interpretation that farmers that This indicates that there are limitations to what a voluntary private sector approach can achieve in terms of improving the uptake of more SI practices. Additionally, if the primary obstacle for farmers to adopt such contracts is maintaining flexibility, similar limitations may apply to voluntary contracting by the public sector through AES, as illustrated by e.g. by Espinosa-Goded et al. 5. Discussion (2010) and Christensen et al. (2011), since these contracts may be perceived as equally restrictive. In order for farmers less willing to engage in SI through these voluntary approaches 9 9 Ecological Economics 211 (2023) 107876 C.S. Weituschat et al. to still implement these practices and improve the overall sustainability of the agri-food system, alternative approaches may thus be necessary, which we will discuss in the concluding section. other hand, will require more efforts and likely alternative weed man agement options. However, almost 40% of farmers in the sample are unlikely to be convinced to adopt SI practices through contract farming. All else equal, it is particularly farmers who are not members of co operatives, are more educated and who generally prefer more flexibility in their sales arrangements that will resist this mechanism to promote practice adoption. If all farmers, and not only some, need to adjust their practices in order to reduce the environmental impacts of the agricul tural sector as a whole, contract farming approaches may not be suffi cient. Instead, policy interventions might be still needed to enhance participation, adoption and diffusion of SI practices. For instance, in centives such as linking subsidies to the adoption of SI practices, particularly through the CAP, may be a mechanism to enhance partici pation, and reach additional farmers. There is indeed potential to couple public interventions with private, supply chain based initiatives, like the one we have investigated in this study. Particularly if public policy mechanisms aim at supporting longer crop rotations, private sector initiatives may also benefit from synergies as farmers could reap benefits through both. However, it is unlikely that private initiatives would cover practices already subsidised through the CAP, and these may thus also function as substitutes. Alternatively, contracting may need to be more participatory to include mutual learning and knowledge exchange. This may overcome barriers other than financial incentives, such as personal preferences or biases farmers may have towards certain practices. Overall, it is likely that no single approach, policy or mechanism can reach all farmers, and that a combination of measures will be necessary to promote SI practices more broadly. Due to the COVID-19 pandemic, we had to adjust the data collection strategy, and while we controlled for that in the analysis, in future research it would be preferable to avoid this. Funding The authors acknowledge support by the EU H2020 project Diver farming—Grant Agreement 728003. 5. Discussion Still, if the experimental design had been too complex to be collected via phone, we would have likely seen a preference for the opt-out alternative for those interviewed by phone (Campbell and Erdem, 2019), but instead we observed that farmers interviewed by phone were less likely to opt out. Unfortunately, our experimental design was not set up to test for interactions between attributes, such as between flower strips as a source of weeds and the use of glyphosate. Further, while the case presented here is exemplary of farmers integrated in industrial value chains, the sample presented here did not aim to be regionally representative given that we targeted this specific group of farmers and needed to access them through channels known to them in order to discuss such business-sensitive issues. We therefore do not aim to generalise to farmers in the region. Also, there is most likely regional specificity. As is the case for finding suitable SI practices for each regional cropping system, farmers’ preferences for practices and contract attributes are also likely to differ by region. It is thus advised that similar studies will be repeated for other locations. It would also be useful to engage in a direct comparative analysis to investigate whether and which farmers prefer to engage with public or value chain partners when it comes to contracting for more sustainable practices, or if contracts with either of these parties is considered equally restrictive. In addition, as L¨apple and Rensburg (2011) suggest, the current degree of diffusion of a practice likely influences who adopts a practice and why. It is therefore advised to observe changes in farmers’ preferences over time, depending on the continued uptake of SI practices (Dessart et al., 2019). Finally, a large majority of farmers in this sample were men. Further research may thus want to oversample for female farmers and farmers of other marginalised groups in order to assess the specific dynamics they may face in contracting for SI. Data availability Data will be made available on request. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. 6. Conclusion This study has highlighted that contract farming is a potential mechanism to support farmers in adopting SI practices. Many farmers in the sample see benefits in contracting for SI, and they are likely to accept legumes in their rotations and flower strips in their fields. Completely banning or strongly limiting glyphosate in their cropping system, on the References empirical study in southern France. Ecol. Econ. 163, 113–125. https://doi.org/ 10.1016/j.ecolecon.2019.03.026. 10.1016/j.ecolecon.2019.03.026. Campbell, D., 2007. Willingness to pay for rural landscape improvements: combining mixed logit and random-effects models. J. Agric. Econ. 58 (3), 467–483. Campbell, D., Erdem, S., 2019. Including opt-out options in discrete choice experiments issues to consider. 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When the COVID-19 pandemic initially reached Italy in the spring of 2020 and several government lockdowns restricted movements and gatherings, the data collection was halted. Due to risk of infection, the initial data collection strategy had to be abandoned. Instead, two different strategies were used between the end of September 2020 and end of February 2021. First, agricultural advisors active in the given value chain and willing to participate were trained as enumerators. The majority of enumerators then used their own network to target farmers participating in the CDM initiative and the Barilla value chain, while one enumerator used the advisory service’s data base of clients to target comparable farmers in the same areas. In order to eliminate additional risk of infection, the enumerators targeted farmers in their network whom they were visiting anyway for other activities and then conducted face-to-face interviews. In this manner, 130 farmers were interviewed. Secondly, whenever farmers could not be visited in person, phone interviews were done in order to avoid a possible selection bias based on availability and willingness for an in-person visit. The hypothetical choice experiment in these phone interviews was visually supported by sending the contract options as pdf to the respondent’s phone. By phone, we reached 114 farmers. The average response rate of this secondary, one-on-one approach was approximately 91.6%. We assume the achievement of such a response rate was due to the enumerators being known contacts to the farmers and thus increasing their willingness to participate. Five observations were incomplete and dropped from the analysis bringing the total from 319 to 314 farmers. 10 Ecological Economics 211 (2023) 107876 C.S. Weituschat et al. C.S. Weituschat et al. Environmental and collective attitudes (6 items) Environmental and collective attitudes (6 items) If I do something for the environment just as a single person, it will have no effect.* Since other farmers already contribute to sustainable crop productions, my contribution is not relevant.* The best way to solve environmental problems is to act collectively. Forming an association with other farmers to contribute to environmental improvement is just a waste of time.* For me, participating in collective actions related to the realization of a sustainable supply chain is important to help the environment. M f il d f i d ld b d f if I ib h li i f i bl l h i If I do something for the environment just as a single person, it will have no effect.* Forming an association with other farmers to contribute to environmental improvement is just a waste of time.* For me, participating in collective actions related to the realization of a sustainable supply chain is important to help the environment. Forming an association with other farmers to contribute to environmental improvement is just a waste of time.* For me, participating in collective actions related to the realization of a sustainable supply chain is important to help t environment. My family and my friends would be proud of me if I contribute to the realization of a sustainable supply chain. *reversed in code Preferences for the sale of soft wheat (4 items) It is important for my farm to fix a minimum reference price for soft wheat before sowing. It is important that quality control systems for my soft wheat are clear and transparent It is important for my farm to compare prices for soft wheat between different buyers before selling it. For me it is a problem if I cannot plan the sale of my soft wheat in advance. Satisfaction with usual wheat buyer in the last three years (4 items) The agreement with my usual buyer gives me a fair price. The agreement with my usual buyer has clear quality requirements. The agreement with my usual buyer is a long term relationship. The agreement with my usual buyer covers at least my cost. It is important for my farm to compare prices for soft wheat between different buyers before selling it. For me it is a problem if I cannot plan the sale of my soft wheat in advance. References Appendix B. Attitudinal scales Below the translated sets of items for the attitudinal scales used in the analysis. 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https://openalex.org/W2108860948	https://europepmc.org/articles/pmc2796013?pdf=render	English	null	Personality Characteristics of Mothers of Children with Attention Deficit Hyperactivity Disorder as Assessed by the Minnesota Multiphasic Personality Inventory	Psychiatry investigation	2,008	cc-by	2,959	online © ML Comm online © ML Comm 0ORIGINAL ARTICLE0 Psychiatry Invest 2008;5:228-231 0ORIGINAL ARTICLE0 Psychiatry Invest 2008;5:228-231 Print ISSN 1738-3684 / On-line ISSN 1976-3026 Personality Characteristics of Mothers of Children with Attention Deficit Hyperactivity Disorder as Assessed by the Minnesota Multiphasic Personality Inventory Soon Jeong Lee, MD, PhD1 Jung-Hwa Kwon, MD, PhD1 Yu Jin Lee, MD, PhD2 Soon Jeong Lee, MD, PhD1 Jung-Hwa Kwon, MD, PhD1 Yu Jin Lee, MD, PhD2 1Department of Psychiatry, Seoul Metropolitan Eunpyong Hospital, Seoul, 2Department of Psychiatry, Gachon University Gil Hospital, Incheon, Korea ObjectiveᄏThe current study investigated the personality characteristics of mothers of children with attention deficit hyperactivity disorder (ADHD) using the Minnesota Multiphasic Per- sonality Inventory (MMPI). MethodsᄏFifty mothers (average age of 38.1±4.2 years) of children with ADHD not having comorbidity (37 boys, 13 girls; average age of 8.5±1.9 years) and 59 mothers (average age of 38.1±2.7 years) of comparison children (37 boys, 13 girls; average age of 8.1±1.5 years) completed the Korean version of the MMPI. Only mothers whose psychiatric health was ver- ified by the Structured Clinical Interview for axis-I DSM-IV disorders (SCID-IV) were included in current study. ResultsᄏAfter controlling for maternal age, maternal education level, children’s gender, age, and total and verbal intelligence quotient (IQ), the MMPI scores of the mothers of children with ADHD were significantly higher on the depression (D), hysteria (Hy) and psychasthenia (Pt) scales than those of the mothers of children in the comparison group. ResultsᄏAfter controlling for maternal age, maternal education level, children’s gender, age, and total and verbal intelligence quotient (IQ), the MMPI scores of the mothers of children with ADHD were significantly higher on the depression (D), hysteria (Hy) and psychasthenia (Pt) scales than those of the mothers of children in the comparison group. ConclusionᄏThese results suggested that even psychologically healthy mothers of children with ADHD alone might be depressed, histrionic and anxious. KEY WORDS: Attention deficit hyperactivity disorder, Mother, Personality, Minnesota Multiphasic Personality Inventory. Psychiatry Invest 2008;5:228-231 Correspondence Yu Jin Lee, MD, PhD Department of Psychiatry, Gachon University Gil Hospital, 1198 Guwol-dong, Namdong-gu, Incheon 405-760, Korea Tel +82-32-460-3696 Fax +82-32-460-3434 E-mail leeyj1203@gmail.com Copyright ⓒ 2008 Official Journal of Korean Neuropsychiatric Association Introduction A number of previous studies have noted an increased incidence of psychopatho- logy and personality disorder in parents of children with attention deficit hyperac- tivity disorder (ADHD).1-3 However, they demonstrated that parents of children with ADHD comorbid with conduct disorder and oppositional defiant disorder were more likely to have mood disorders, anxiety disorders, antisocial personalities and engage in substance abuse.1 In another study, conduct disorder but not ADHD was reported to be linked to parental psychopathology by a structured psychiatric interview.4 Several previous studies used the Minnesota Multiphasic Personality Inventory (MMPI)5 to investigate the personalities of parents of children with ADHD, but their findings were inconsistent.6-8 Mothers of children with ADHD were previously reported to score the highest on the depression (D), hysteria (Hy) and psychopathic deviate (Pd) scales of the MMPI, but a comparison group was not included.6 Lahey (1989) noted that mothers of children with ADHD without conduct disorder did not score higher on any scale of the MMPI than the mothers of children in the comparison group, but suggested that coexisting conduct disorder could predict maternal personality disturbances.7 In another study, parents of children with ADHD showed higher Pd scores on their MMPI Correspondence Yu Jin Lee, MD, PhD Department of Psychiatry, Gachon University Gil Hospital, 1198 Guwol-dong, Namdong-gu, Incheon 405-760, Korea Tel +82-32-460-3696 Fax +82-32-460-3434 E-mail leeyj1203@gmail.com 228 Copyright ⓒ 2008 Official Journal of Korean Neuropsychiatric Association 228 www.psychiatryinvestigation.org SJ Lee et al. profiles than those in the community comparison group.8 These inconsistencies might result from the high preval- ence of psychiatric illnesses among parents of children with ADHD and the lack of homogenous group of ADHD patients. To date, no study has measured the MMPI profiles of psychologically healthy mothers of children with ADHD not comorbid with conduct disorder or oppositional defiant disorder. In the current study, we aimed to examine the per- sonality characteristics of mothers of children with ADHD using the MMPI. of 8.5±1.9 years) and 59 mothers (average age of 38.1± 2.7 years) of comparison children (37 boys, 13 girls; ave- rage age of 8.1±1.5 years) completed the Korean ver- sion of the MMPI. The MMPI is one of the most freque- ntly used tests for personality assessment. Introduction It was developed in 1943 as a screening tool for psychopathology and as- sessment of personality.5 It consists of 566 self-report true/ false questions, which are scored for 8 clinical scales of psychopathology, 2 scales of personality dimensions i.e. masculinity-femininity (Mf) and social introversion (Si), and 3 validity scales. SPSS version 12.0 software was used for the statistical analysis. Result Fifty-six biological mothers of outpatients in the Seoul Metropolitan Eunpyeong Hospital with Diagnostic and Stati- stical Manual-fourth edition (DSM-IV)9-diagnosed ADHD and 63 biological mothers of comparison children who re- sponded to advertisements for this study were recruited. Children with ADHD comorbid with other psychiatric di- sorders, including conduct disorder and oppositional de- fiant disorder, by semi-structured interviews and medical record reviews were not included in the present study. Comparison children had no psychiatric history, no cur- rent psychiatric symptoms and no current behavioral issues based on a semi-structured interview with DSM-IV criteria. The Structured Clinical Interview for axis-I DSM-IV di- sorders (SCID-IV)10 was administered to all mothers in order to evaluate their mental health. Due to the presence of current psychiatric disorders by SCID-IV or previous psychiatric history, six of the mothers in the ADHD group (dysthymia: 1, panic disorder: 1, major depressive disor- der: 2, ADHD history: 1, and conduct disorder history: 1) and 4 of the mothers in the comparison group (dysthymia: 1, panic disorder: 1, generalized anxiety disorder: 1, and alcohol abuse history: 1) were excluded. Children with in- telligence quotient (IQ) scores under 75 measured by the Korean Educational Development Institute-Wechsler In- telligence Scale for Children (KEDI-WISC) were exclu- ded. None of the children had IQs below 80. The mean scores on all of the MMPI scales were within the normal limits for both groups of participants. There were no significant differences in maternal age, maternal educational level, or children’s age between the ADHD and comparison groups (Independent t-test; p=0.933, p= 0.773 and p=0.201 respectively, Table 1). There was no significant difference in the gender of the children between the two groups (Chi-square test; p=0.224, Table 1). The total and verbal IQ scores of the children with ADHD were significantly lower than those of the children in the comparison group (Independent t-test; p=0.023 and p= 0.003 respectively; Table 1). After controlling for ma- ternal age, maternal education, children’s age, gender, and total and verbal IQ, the mothers of children with ADHD scored higher on the D, Hy and Pt scales (p=0.027, p= 0.007 and p= 0.037 respectively, Table 2). www.psychiatryinvestigation.org 229 rental personality disorder.7 In the current study, the mothers of children with ADHD scored higher on the following MMPI scales: D, which measures depression; Hy, which measures hysterical re- actions and denial of stressful situations; and Pt, which measures anxiety, than those of the comparison children. These findings support those of previous studies report- ing that mothers of children with ADHD tended to be more depressed, self-blaming and socially isolated.11 They are also consistent with a previous finding demonstrat- ing an increased risk of mood and anxiety disorders in parents of children with ADHD.12 However, Lahey et al. did not find higher MMPI scale scores in mothers of chil- dren with ADHD, as compared to those in the control group.7 We found strong statistical significance for the hi- gher Hy scores on the MMPI profiles of mothers of chil- dren with ADHD. However, a prior study, which assessed psychopathology using a structured interview, suggested a relationship between maternal hysteria and conduct di- sorder, but not ADHD.4 An objective personality test, such as the MMPI, might be more sensitive to subclinical per- sonality traits than the formal diagnosis of a disorder by clinical interview.7 These discrepancies could be due to the strength of the current study, which had a larger sam- ple size, was exclusive to mothers with psychiatric disor- ders, and controlled for more possible confounding fac- tors in the statistical analysis. Parents play an important role in delivering the proper treatment to children with ADHD. It would not be sur- prising that maternal depression is related to their poor parenting behavior.17 Parental depression, marital distress and parental skills were noted to have a negative effect on treatment compliance in children with ADHD.18 The- refore, the evaluation and proper psychiatric interventions for mothers of children with ADHD could be crucial fac- tors in the treatment process, and an understanding of maternal personality characteristics might be helpful for improving the prognosis of children with ADHD. The present study has several limitations, including its relatively small sample size and the lack of a parametric assessment investigating the severity of psychopathology in the mothers or children. As conduct behaviors in ADHD could become an issue in later in life, the exclusion of children with ADHD and behavioral problems might not be perfect. In addition, the total and verbal IQ scores of children with ADHD were significantly lower than those of the comparison children. Discussion To the best of our knowledge, this is the first study to assess the personality characteristics of mothers whose psychiatric health was verified using the SCID-IV. We also did not enroll children with ADHD who had other psychiatric comorbidities in the current study based on previous findings suggesting that there may be a strong association between coexisting conduct disorder and pa- Finally, fifty mothers (average age of 38.1±4.2 years) of children with ADHD (37 boys, 13 girls; average age TABLE 1. Comparison of demographic findings between the ADHD and comparison groups ADHD group (N=50) Comparison group (N=59) t p value Maternal age (years) 038.1±04.2 038.1±02.7 0.084 0.933 Maternal education (years) 013.5±02.2 013.6±02.3 -0.290 0.773 Gender (No. children)† 37 boys/13 girls 37 boys/12 girls 0.224 Age (years, children) 008.5±01.9 008.1±01.5 1.288 0.201 Total IQ (score, children) 105.2±13.8 111.5±14.5 -2.307 0.023* Performance 106.7±15.7 110.9±14.4 -1.430 0.156 Verbal 102.6±12.1 110.5±15.0 -3.012 0.003* Independent t-test, p<0.05, †Chi-square test. ADHD: attention deficit hyperactivity disorder, IQ: intelligence quotient TABLE 1. Comparison of demographic findings between the ADHD and comparison groups ADHD group (N=50) Comparison group (N=59) TABLE 1. Comparison of demographic findings between the ADHD and comparison groups Personality of Mothers of ADHD Children TABLE 2. Comparison of the MMPI scores of mothers of children in the ADHD and comparison groups ADHD group (N=50) Comparison group (N=59) F p value L 49.8±09.7 50.5±10.8 0.188 0.665 F 46.4±09.9 43.8±08.1 3.277 0.073 K 58.3±11.6 58.8±10.7 0.161 0.689 Hs 54.8±11.1 52.4±08.5 0.783 0.378 D 56.4±11.9 50.8±11.6 5.057 0.027 Hy 56.5±11.0 50.9±09.9 7.670 0.007** Pd 56.6±10.4 52.1±11.2 3.667 0.058 Mf 45.7±09.5 48.2±08.5 3.383 0.069 Pa 49.9±09.3 46.4±09.9 3.643 0.059 Pt 52.7±10.2 48.7±10.0 4.452 0.037* Sc 49.1±10.6 46.0±09.6 2.361 0.128 Ma 45.4±07.7 45.4±08.0 0.149 0.701 Si 49.3±09.3 46.4±10.8 2.329 0.130 ANCOVA controlling for maternal age, children’s age, gender, total IQ and verbal IQ by general linear model in SPSS 12.0, p< 0.05, *p<0.01. MMPI: Minnesota Multiphasic Personality Inven- tory, ADHD: attention deficit hyperactivity disorder, L: lie, F: in- frequency, K: defensiveness, Hs: hypochondriasis, D: depression, Hy: hysteria, Pd: psychopathic deviate, Mf: masculinity-femini- nity, Pa: paranoia, Pt: psychasthenia, Sc: schizophrenia, Ma: hypomania, Si: social introversion TABLE 2. Comparison of the MMPI scores of mothers of children in the ADHD and comparison groups ADHD did not score significantly high on the Pd scale of the MMPI. Discussion A higher Pd score was a consistent finding in two previous family studies that measured the parental MMPIs of children with ADHD.6,8 A plausible explanation for this discrepancy might be the exclusion of children with ADHD comorbid with conduct disorder and oppo- sitional defiant disorder in the current study. The notion that an antisocial personality in men and hysteria in women may share the same origin,13 rather than elevated Pd score, would be another plausible explanation for the higher Hy scores in the current study, which included only female subjects. Many aspects of personality are known to be under pa- rtial genetic influence. In a prior study, the heritability es- timates of MMPI scores were from 0.26 to 0.61.14 The- refore, the higher D and Pt scores on the maternal MMPI profiles in the current study might support previous notion that children with ADHD were highly likely to have com- orbid anxiety and depression and that they have more diffi- culties in their social relationships.15 In addition, ADHD it- self has been suggested to have a large genetic component.16 However, a non-genetic understanding of these findings is possible. The MMPI scores could be more state-depend- ent than trait-dependent in the individual’s lifetime.8 Our findings of more depression, hysteria and anxiety on the MMPI could reflect the maternal psychological burden of raising a child with a chronic behavioral disorder rather than a suggestion of personality characteristics with a genetic predisposition to ADHD Many aspects of personality are known to be under pa- rtial genetic influence. In a prior study, the heritability es- timates of MMPI scores were from 0.26 to 0.61.14 The- refore, the higher D and Pt scores on the maternal MMPI profiles in the current study might support previous notion that children with ADHD were highly likely to have com- orbid anxiety and depression and that they have more diffi- culties in their social relationships.15 In addition, ADHD it- self has been suggested to have a large genetic component.16 However, a non-genetic understanding of these findings is possible. The MMPI scores could be more state-depend- ent than trait-dependent in the individual’s lifetime.8 Our findings of more depression, hysteria and anxiety on the MMPI could reflect the maternal psychological burden of raising a child with a chronic behavioral disorder rather than a suggestion of personality characteristics with a genetic predisposition to ADHD. 230 Psychiatry Invest 2008;5:228-231 rental personality disorder.7 This finding could support the previous findings that the mean IQ scores of children with ADHD were lower than those of normal controls and that the IQ scores correlated negatively with ADHD In the current study, the mothers of the children with 230 Psychiatry Invest 2008;5:228-231 SJ Lee et al. of offspring with childhood-onset schizophrenia or ADHD. Behav Genet 2005;35:159-175. symptoms.19,20 To rule out the potential effect of the child’s intelligence on the MMPI profile of the mother, we controlled for total and verbal IQ score as a confound- ing factor in the statistical analysis. of offspring with childhood-onset schizophrenia or ADHD. Behav Genet 2005;35:159-175. 9. American Psychiatric Association. Diagnostic and Statistical Manual of Mental Disorders fourth edition (DSM-IV). Washington, DC: American Psychiatric Association; 1994. 10. First MB, Spitzer RL, Gibbon M, Willams JB. Structured Clinical In- terview for Axis I DSM-IV Disorders. New York: Biometrics Research; 1994. In conclusion, these results suggested that even psy- chologically healthy mothers of children with ADHD alone might have a characteristic personality, which was observed to be more depressed, more histrionic and more anxious. 11. Mash EJ, Johnston C. Determinants of parenting stress: illustrations from families of hyperactive children and families of physically abused children. J Clin Child Psychol 1990;19:313-328. children. J Clin Child Psychol 1990;19:313-328. 12. Nigg JT, Hinshaw SP. Parent personality traits and psychopathology associated with antisocial behaviors in childhood attention-deficit hy- peractivity disorder. J Child Psychol Psychiatry 1998;39:145-159. www.psychiatryinvestigation.org 231 REFERENCES peractivity disorder. J Child Psychol Psychiatry 1998;39:145-159. 13. Cloninger CR, Guze SB. Psychiatric illness and female criminality: the role of sociopathy and hysteria in the antisocial woman. Am J Psychi- atry 1970;127:303-311. 1. Chronis AM, Lahey BB, Pelham WE Jr, Kipp HL, Baumann BL, Lee SS. Psychopathology and substance abuse in parents of young children with attention-deficit/hyperactivity disorder. J Am Acad Child Adolesc Psychiatry 2003;42:1424-1432. 14. DiLalla DL, Carey G, Gottesman II, Bouchard TJ Jr. Heritability of MMPI personality indicators of psychopathology in twins reared apart. J Abnorm Psychol 1996;105:491-499. 2. Pfiffner LJ, McBurnett K, Rathouz PJ, Judice S. Family correlates of oppositional and conduct disorders in children with attention deficit/ hyperactivity disorder. J Abnorm Child Psychol 2005;33:551-563. 15. Pliszka SR. Psychiatric comorbidities in children with attention deficit hyperactivity disorder: implications for management. Paediatr Drugs 2003;5:741-750. 3. Johnston C, Mash EJ. Families of children with attention-deficit/hyper- activity disorder: review and recommendations for future research. Clin Child Fam Psychol Rev 2001;4:183-207. 16. Elia J, Devoto M. ADHD genetics: 2007 update. Curr Psychiatry Rep 2007;9:434-439. 4. Lahey BB, Piacentini JC, McBurnett K, Stone P, Hartdagen S, Hynd G. Psychopathology in the parents of children with conduct disorder and hyperactivity. J Am Acad Child Adolesc Psychiatry 1988;27:163-170. 17. Goodman SH. Depression in mothers. Annu Rev Clin Psychol 2007; 3:107-135. 18. Chronis AM, Lahey BB, Pelham WE Jr, Williams SH, Baumann BL, Kipp H, et al. Maternal depression and early positive parenting predict future conduct problems in young children with attention-deficit/hy- peractivity disorder. Dev Psychol 2007;43:70-82. 5. Hathaway SR, McKinley JC. Minnesota Multiphasic Personality In- ventory. New York: Psychological Corporation; 1943. 6. Firestone P, Witt JE. Characteristics of families completing and prema- turely discontinuing a behavioral parent-training program. J Pediatr Psychol 1982;7:209-222. 19. Mariani MA, Barkley RA. Neuropsychological and academic functio- ning in preschool boys with attention deficit hyperactivity disorder. Dev Neuropsychol 1997;13:111-129. 7. Lahey BB, Russo MF, Walker JL, Piacentini JC. Personality charac- teristics of the mothers of children with disruptive behavior disorders. J Consult Clin Psychol 1989;57:512-515. 20. Simonoff E, Pickles A, Wood N, Gringras P, Chadwick O. ADHD sy- mptoms in children with mild intellectual disability. J Am Acad Child Adolesc Psychiatry 2007;46:591-600. 8. Subotnik KL, Asarnow RF, Nuechterlein KH, Fogelson DL, Thorpe TI, Payne DL, et al. MMPI vulnerability indicators for schizophrenia and attention deficit disorder: UCLA family study of biological parents
W1963577933.txt	https://www.scielo.br/j/rsp/a/fFc9gx5cxNYfQbpVXzjvQWP/?lang=pt&format=pdf	en		Prevalência de marcadores sorológicos de hepatite B numa pequena comunidade rural do Estado de São Paulo, Brasil	Revista de saúde pública/Revista de Saúde Pública	1,992	cc-by	3,179	Prevalência de marcadores sorológicos de hepatite B numa pequena comunidade rural do Estado de São Paulo, Brasil* Prevalence of serological markers of hepatitis B in a small rural community of S. Paulo State, Brazil Afonso D.C, Passos, Uilho A. Gomes, José F. de C. Figueiredo *, Margarida M. P. do Nascimento, Jacqueline M. de Oliveira*, Ana M. C. Gaspar, Clara F. T. Yoshida** PASSOS, A. D. C. et al. Prevalência de marcadores sorológicos de hepatite B numa pequena comunidade rural do Estado de São Paulo, Brasil. Rev. Saúde públ, S. Paulo, 26: 119-24, 1992. Foi realizado estudo epidemiológico com vistas a determinar a prevalência de marcadores sorológicos de hepatite B na população de um pequeno município, de características rurais, do Estado de São Paulo. Observou-se prevalência total de marcadores igual a 7,74%, com valores de HBsAg, anti-HBs e anti-HBc, respectivamente iguais a 0,10%, 1,69% e 7,64%. Ressalta-se a importância da determinação do anti-HBc em estudos epidemiológicos, bem como discute-se a relevância de se comparar a reduzida circulação viral, observada na área, com as elevadas prevalências verificadas em outras regiões, buscando assim levantar hipóteses acerca de mecanismos alternativos de transmissão. Descritores: Hepatite B, epidemiologia. Estudos de prevalência. População rural. Introdução Entre os inúmeros aspectos ainda não totalmente entendidos na história natural da hepatite B, destaca-se a grande variação existente na distribuição de marcadores específicos em populações diversas, às vezes semelhantes entre si no que diz respeito a características gerais6. A tentativa de aclarar estas variações exige a realização de estudos de campo em larga escala, abrangendo populações variadas em sua composição e características sociais e em diferentes situações geográficas19. Estudos de prevalência em amostras populacionais representativas, contudo, praticamente inexistem no Brasil, onde a quase totalidade dos dados disponíveis se refere a grupos restritos 1,8,10,13,16,17,20,23 . Dificulta-se com isso não apenas a generalização dos acha- * Parte da tese de doutorado apresentada ao Departamento de Medicina Social da Faculdade de Medicina de Ribeirão Preto da Universidade de São Paulo, 1991. Departamento de Medicina Social da Faculdade de Medicina de Ribeirão Preto - Ribeirão Prelo, SP - Brasil * Departamento de Clínica Médica da Faculdade de Medicina de Ribeirão Preto - Ribeirão Prelo, SP - Brasil **** Centro de Referência Nacional para Hepatites Virais. FIOCRUZ. Rio de Janeiro, RJ - Brasil Separatas/Reprints; A. D. C. Passos - Av. Bandeirantes, 3900 14049-900 - Ribeirão Preto, SP - Brasil Publicação financiada pela FAPESP. Processo Saúde Coletiva 91/4994-0 dos, mas também a possibilidade de que os mesmos possam ser comparados. A necessidade de estudo de prevalência faz-se sentir de modo particular em pequenas comunidades situadas em regiões afastadas dos grandes centros, a respeito das quais muito pouco se conhece no que se refere à situação epidemiológica da hepatite B. Visando caracterizar alguns aspectos da história natural desta doença, em tais situações, foi realizado estudo de campo englobando cerca de 70% da população de Cássia dos Coqueiros, um pequeno município de 2.800 habitantes, com características rurais, situado a 80 Km de Ribeirão Preto, no Estado de São Paulo. Um dos objetivos do estudo consistiu na determinação da prevalência de marcadores sorológicos de hepatite B nos habitantes daquela comunidade, constituindo os seus achados o motivo do presente trabalho. Material e Método Toda a população do Município de Cássia dos Coqueiros foi inicialmente visitada em agosto/ setembro de 1986, ocasião cm que se procedeu a uma atualização do cadastro de moradores, e em que inúmeras informações concernentes às residências foram coletadas. No período de outubro de 1986 a maio de 1990 os indivíduos cadastrados e com idade igual ou superior a um ano foram chamados ao Centro de Saúde daquele Município (no caso de moradores urbanos) ou re-visitados (habitantes rurais) para que, caso desejassem, fossem incluídos no estudo após tomarem conhecimento dos seus objetivos e dos procedimentos que seriam adotados. A cada indivíduo incluído na investigação foi aplicado um questionário específico, visando levantar diferentes fatores de risco para hepatite B. Simultaneamente, foi coletada uma amostra de 10 ml de sangue por punção venosa cubital. O soro de cada indivíduo foi submetido a exames imunoenzimáticos para detecção de HBsAg, anti-HBs e antiHBc total, utilizando-se para isso de reagentes produzidos e cedidos pelo Centro de Referência Nacional para Hepatites Virais (CRNHV), da Fundação Instituto Oswaldo Cruz. A detecção de HBsAg e anti-HBs foi feita por meio de ensaio imunoenzimático indireto7,22, utilizando-se para leitura um espectrofotômetro Minireader 2. O ponto de corte foi definido multiplicando-se a média das densidades ópticas dos controles negativos por 2,1. Assim, foram consideradas, respectivamente como positivas e negativas, as amostras que se situassem acima e abaixo do valor assim obtido, A determinação do anti-HBc total foi feita por meio de ensaio imunoenzimático de inibição, utilizando-se para leitura dos resultados um espectrofotômetro Minireader 2.O ponto de corte foi calculado pela fórmula: onde: cont. pos. = média da densidade óptica dos controles positivos. cont. neg. = média da densidade óptica dos controles negativos. Os exames de detecção de HBsAg e anti-HBs foram realizados por uma única pessoa, a qual realiza, de rotina estes procedimentos no Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto. Os exames de detecção de anti-HBc total foram realizados por uma funcionária do CRNHV. Todos os exames com resultado acima do "cutoff", definido para a reação, foram repetidos, considerando-se como positivas aquelas amostras que no segundo exame apresentaram também um resultado superior ao ponto de corte. A análise estatística foi realizada mediante a aplicação do teste qui-quadrado, fixando-se o valor de alfa = 0,05 como limite de significância para rejeição da hipótese de nulidade. Resultados A Tabela 1 mostra a distribuição dos indivíduos estudados e da prevalência de marcadores de hepatite B segundo o sexo e a idade. A prevalência de positivos revela padrões semelhantes nos dois sexos até a faixa dos 35 anos de idade (Figura). Muito embora ocorra, após esta faixa etária, tendência de predomínio entre os homens, a qual se acentua mais nitidamente acima dos 65 anos, esta diferença não se revelou estatisticamente significativa - [x2 (8 gl)= 10,66; p = 0,2217]. A Tabela 2 mostra a distribuição dos marcadores de hepatite B pesquisados, de acordo com a faixa etária de ocorrência. Apenas dois portadores crônicos de HBsAg foram detectados, um com 38 e outro com 43 anos de idade, resultando numa prevalência total de 0,10% para este marcador. A positividade para o anti-HBs começa a se fazer presente no grupo de 10 a 15 anos, aumentando progressivamente nas idades mais elevadas e perfazendo uma prevalência total de 1,69%. As maiores prevalências são observadas para o anti-HBc total, já presente no grupo de 1 a 5 anos de idade e com tendência claramente ascendente nos grupos etários mais elevados, chegando a atingir um valor máximo de 18,9% nos indivíduos acima de 65 anos. A prevalência global deste marcador atinge um valor muito próximo daquele observado para a somatória de todos os marcadores sorológicos presentes, combinados ou isolados (7,69% e 7,74%, respectivamente). A análise estatística mostra diferenças significantes nas prevalências de marcadores nas diversas faixas etárias - [x2 (8 gl) = 33,20; p = 0,0001]. Embora não representado na tabela, merece ser destacado o fato de que 32 entre os 33 indivíduos com anti-HBs positivo também apresentaram positividade para o anti-HBc. Discussão A positividade dos diferentes marcadores, nas amostras examinadas, revela-se bastante diferenciada do ponto de vista quantitativo (Tabela 2). Enquanto somente dois indivíduos apresentaramse como portadores do antígeno HBsAg, a positividade para o seu anticorpo específico fez-se presente em 33 pessoas. Todavia, o estudo da presença do anti-HBc acrescentou outras 110 amostras à lista dos positivos, atingindo-se assim o total de 151 pessoas cm que um ou mais marcadores foram detectados. Uma primeira observação que se impõe, portanto, em trabalhos de determinação de prevalência, reside na importância de detecção do anti-HBc. Com efeito o acréscimo deste marcador permitiu ampliar de maneira considerável o conhecimento da situação da hepatite B na população de estudo, fornecendo uma imagem certamente muito mais aproximada de transmissão e circu- lação do agente infeccioso na comunidade. Deve ser lembrado que foram consideradas como positivas apenas as amostras que confirmaram a sua positividade por ocasião de um segundo exame, realizado em todas as que inicialmente mostraram resultado acima do ponto de corte. Este cuidado foi tomado com a finalidade de se evitar ao máximo a ocorrência de falsos positivos, possibilidade presente de modo especial na determinação do anti-corpo anti-HBc21. Uma segunda observação relevante que pode ser feita a partir destes dados diz respeito aos valores consideravelmente baixos de prevalência encontrados em Cássia dos Coqueiros. Critérios internacionalmente aceitos, para definição de padrões de distribuição de hepatite B, consideram como de baixa prevalência áreas que apresentem as seguintes características: a) prevalência de HBsAg entre 0,1% e 0,5%; b) prevalência de antiHBs entre 4% e 6% e c) infecções neonatais e na infância ausentes ou raras21. Assim, as cifras obtidas em Cássia dos Coqueiros são comparáveis às verificadas fora das populações de risco da América do Norte, Europa Ocidental, Austrália e parte temperada da América do Sul, consideradas como de baixa endemicidade para a doença21. Nessas regiões, a história natural da hepatite B apresenta um padrão de circulação onde se destaca a reduzidíssima ou inexistente transmissão vertical, tornando a distribuição da doença um fenômeno esporádico, associado quase que exclusivamente à transmissão por via sexual ou pelo uso de seringas/agulhas contaminadas. Conseqüência direta da falta de um considerável reservatório de infectados na população, a raridade ou inexistência de transmissão vertical ocasiona uma distribuição etária característica, com uma positividade que se revela escassa em idades precoces e tende a uma elevação lenta e gradual à medida que aumenta a faixa etária14. Este padrão se observa em ambos os sexos na população do presente estudo (Tabela 1 e Figura). As baixas prevalências de hepatite B fora das tradicionais populações de risco da América do Norte, Europa Ocidental, Austrália e parte temperada da América do Sul têm sido associadas às melhores condições sócio-econômicas das populações dessas partes do mundo. Considerando-se que essas condições devam ser bastante superiores às verificadas na área do presente estudo, uma questão que se levanta diz respeito aos mecanismos pelos quais os níveis de endemicidade permanecem semelhantes em regiões tão diferenciadas quanto às condições gerais de vida. Uma possível explicação para a baixa endemicidade em Cássia dos Coqueiros poderia, talvez, ser encontrada em certas características inerentes às peque- nas comunidades rurais do interior brasileiro, tais como o relativo isolamento geográfico e a predominância de padrões de comportamento mais tradicionais e conservadores. Claro está que apenas isto não serve como explicação totalmente satisfatória, uma vez que elevados níveis de endemicidade também são descritos em pequenas comunidades relativamente isoladas do próprio interior brasileiro, como testemunham os achados entre tribos indígenas e populações nativas da Amazônia 2 , 3 , 4 , 5 , 9 , 1 1 , 1 2 . É possível que a condição verificada em Cássia dos Coqueiros se deva à combinação de suas características rurais com o fato de estar localizada numa macrorregião geográfica (Sudeste) onde a prevalência da doença se situa em níveis reduzidos, comparativamente a áreas da Amazônia brasileira13. Achados recentes dando conta de elevadas prevalências em populações relativamente isoladas de Iguape e Cananéia,15,18 contradizem, à primeira vista, esta explicação. Entretanto, deve ser lembrado que a peculiaridade de uma nítida sobreposição verificada nas distribuições de marcadores de hepatite B e de arboviroses, nessas regiões do Vale do rio Ribeira, sugere que mecanismos de transmissão comuns a estas diferentes doenças possam se fazer presentes na área, levantando inclusive a hipótese de envolvimento de mosquitos com atividade antropofílica15,18. O presente estudo lança alguma luz sobre a história natural da hepatite B numa situação particular, representada por um pequeno município de características rurais, relativamente isolado, do Estado de São Paulo. A reduzida circulação do vírus, aqui verificada, pode vir a servir como um importante elemento de comparação para outras investigações. Assim, o confronto das semelhanças e/ou diferenças entre a população de Cássia dos Coqueiros e as de outros estudos, envolvendo comunidades rurais, pode levantar pistas acerca de possíveis determinantes dos níveis diferenciados de transmissão observados em comunidades variadas. Como exemplo, o flagrante contraste entre a baixa prevalência obtida no presente trabalho e os elevados valores observados em áreas rurais do Vale do Ribeira15,18 reforçam a hipótese do envolvimento de insetos antropofílicos na transmissão da hepatite B, nessa região. Com efeito, uma diferença que se destaca entre estas duas áreas rurais é a presença de florestas, abundantes no Vale do Ribeira e ausentes em Cássia dos Coqueiros, acarretando assim densidades desiguais de insetos. Obviamente, comparações deste tipo não definem associações, exigindo-se para isso um aprofundamento nos estudos epidemiológicos que os levem além da mera determinação e comparação de prevalência totais. Isto porque a simples análise dos valores de prevalência referentes a toda uma população pode esconder variações internas importantes, eventualmente existentes em segmentos de uma mesma comunidade que difiram entre si no que diz respeito a variáveis tais como a situação sócio-econômica, procedência, hábitos de vida e escolaridade, entre outras. Tais comparações, entretanto, prestam-se a levantar pistas acerca de diferentes fatores passíveis de serem incriminados como envolvidos na transmissão da hepatite B. Deve-se considerar ainda que a prevalência aqui encontrada talvez possa refletir o que se passa em situações similares, contribuindo para, eventualmente, reduzir o desconhecimento sobre a epidemiologia da hepatite B em comunidades semelhantes a do presente estudo, certamente comuns no interior brasileiro. PASSOS, A. D. C. et al. [Prevalence of serological markers of hepatitis B in a small rural community of S. Paulo State, Brazil]. Rev. Saúde públ., S. Paulo, 26: 119-24, 1992. Prevalence of three hepatitis B markers was measured by immunoassay techniques in small rural community of the State of S. Paulo, Brazil. Total prevalence was 7.74%, corresponding to values of 0.10%, 1.69% and 7.74% for HBsAg, anti-HBs and anti-HBc, respectively. The importance of anti-HBc determination in such studies is stressed. Comparisons between the low viral circulation observed in this area and high prevalences described in other rural communities may contribute to the raising of new hypothesis concerning alternative transmission mechanisms of hepatitis B. Keywords: Hepatitis B, epidemiology, prevalence studies. Rural population. Referências Bibliográficas 1. ABUZWAIDA, A. R. N.; SIDONI, M.; YOSHIDA, C. F. T.; SCHATZMAYR, H. G. Seroepidemiology of hepatitis A and B in two urban communities of Rio de Janeiro. Rev. Inst. Med. trop. S. Paulo, 29: 219-23, 1987. 2. ALECRIM, W. D.; MARREIROS, L. S.; ALECRIM, M. G. C.; SANTOS, I. K. F. M. Inquérito sobre presença de HBsAg em habitantes de Lábrea, Amazonas. [Carta ao Editor]. Rev. Soc. bras. Med. trop., 19: 58-9, 1986. 3. BENSABATH, G. & BOSHELL, J. Presença do antígeno "Austrália" (Au) em populações do interior do Estado do Amazonas, Brasil. Rev. Inst. Med. trop. S. Paulo, 15: 284-8, 1973. 4. BENSABATH, G. & DIAS, L. B. Hepatite de Lábrea (febre negra de Lábrea) e outras hepatites fulminantes em Sena Madureira, Acre e Boca do Acre, Amazonas, Brasil. Rev. Inst. Med. trop. S. Paulo, 25: 182-94, 1983. 5. BENSABATH, G.; HADLER, S. C; SOARES, M.e P.; FIELDS, H.; MAYNARD, J. E. Características serologicas y epidemiologicas de la hepatitis virica aguda en la cuenca amazonica del Brasil. Bol. Ofic. sanit. panamer., 103:351-62,1987. 6. BRABIN, L. & BRABIN, B. Cultural factors and transmisson of hepatitis B virus. Amer. J. Epidem., 122: 725-30, 1985. 7. CAMARGO, I. F.; GASPAR, A. M. C; YOSHIDA, C. F. T. Comparative Elisa reagents for detection of hepatitis B surface antigen (HBsAg). Mem. Inst. Oswaldo Cruz, 82: 181-7,1987. 8. FERRAZ, M. L. G.; VILELA, M. P.; SILVA, A. E.; PAIVA, E. R. Marcadores sorológicos do vírus da hepatite B e anticorpo delta em homossexuais masculinos brasileiros. Rev. paul. Med., 103: 228-30, 1985 9. FIGUEIREDO-MENDES, T. A Amazônia Brasileira e a hepatite B. [Editorial]. Mod. Hepatolol., 14(1): 1-2, 1989. 10. FOCACCIA, R.; VERONESI, R.; TAKEDA, A; BAZONE, J. R. C.; RODRIGUES, E.; MAZZA, C. C; KIMURA, R. T.; FELDMAN, C. Prevalência do antígeno de superfície do vírus da hepatite B (HBsAg) e seu anticorpo (anti-HBs) em pacientes internados em dois hospitais psiquiátricos. Rev. Inst. Med. trop. S. Paulo, 24: 385-7,1982. 11. FONSECA, J. C. F. Hepatite B no Estado do Amazonas. Mod. Hepatol., 14(1): 33-5, 1989. 12. FONSECA, J. C. F. & TAVARES, A. M. Estudo dos marcadores sorológicos do vírus da hepatite B (VHB) em área de ocorrência da hepatite de Lábrea (Febre Negra), Codajás, AM, Brasil. [Cartas ao editor]. Rev. Soc. bras. Med. trop., 19: 117, 1986. 13. HADLER, S. C.; FAY, O. H.; PINHEIRO, F.; MAYNARD, J. E. La hepatitis en las Americas: informe del grupo colaborador de la OPS. Bol. Ofici. sanit. panamer., 103: 185-209, 1987. 14. HEPATITIS virica en la region. Bol. Ofic. sanit. panamer., 100:330-7, 1986. 15. IVERSSON, L. B.; GRANATO, C. F. H.; TRAVASSOS DA ROSA, A.; PANNUTI, C. S. Relationship between the prevalence of antibodies to hepatitis B core antigen and arbovirus in fishermen from the Ribeira Valley, Brasil. Rev. Inst. Med. trop. S. Paulo, 32: 215-20,1990. 16. MAZZUR, S.; NATO, N.; FANG, C; BASTIAANS, M. J.; MOLINARIS, J. L.; BALCASER, M.; BEKER, G. S.; BRUNINGS, E. A.; CAMERON, A. R. E.; FARREL, V.; FAY, O. H.; LABRADOR-GONZALEZ, G.; GONZALEZ, L. G.; GUTIERREZ, A.; JARAMILLO, T. C.; KATZ, R.; LEME LOPEZ, M .B.; LEVY-KOENIG, E.; MORALES AYALA, F; RODRIGUES AMAYA, J.; RODRIGUES-MOYADO, H.; TORRES, R. A.; VELASCO, M. Distribution de marcadores de virus de hepatitis B (VHB) en la sangre de donadores de 13 paises del hemisfério occidental: actas del Taller Latinoamericano de la Cruz Roja sobre Hepatitis B. Bol. Ofici. sanit. panamer., 89: 239-48, 1980. 17. MINCIS, M.; GUIMARÃES, R. X.; NETO, J. F.; NOVO, N. F.;CHECTER, L. Marcadores imunológicos do vírus B da hepatite em alcoólatras e indivíduos normais. Rev. paul. Med., 102: 205-13, 1984. 18. PANNUTI, C. S.; IVERSSON, L. B.; MENDONÇA, J. S.; TRAVASSOS DA ROSA, A. P. A.; GRANATO, C. F. H. Relationship between the prevalence of antibodies to arbovirus and hepatitis B virus in the Vale do Ribeira Region, Brazil. Rev. Inst. Med. trop. S. Paulo, 31: 1039,1989. 19. PASSOS, A. D. C. Estudo epidemiológico sobre hepatite B em Cássia dos Coqueiros, SP, Brasil. Ribeirão Preto, 1991. [Tese de Doutorado - Faculdade de Medicina de Ribeirão Preto da USP]. 20. STRAUS, E.; MAFFEI-JR, R. A.; SÁ, M. F. G.; DUTRA, A. P.; TAKEDA, A. K. Incidência de HBsAg e antiHBs. Freqüência do antígeno de superfície da hepatite B e de seus anticorpos detectados por radioimunoensaio em pessoal hospitalar. Rev. Inst. Med. trop. S. Paulo, 25: 246-53, 1983. 21. U. S. DEPARTMENT OF HEALTH AND HUMAN SERVICES. Public Health Service. Centers for Disease Control. Hepatitis Surveillance. Atlanta, Ga, 1989. (Report, 52). 22. VOLLER, A.; BARTLETT, A.; BIDWELL, D. E. Enzyme immunoassays with special reference to Elisa techniques. J. clin. Pathol., 31: 507-20, 1978. 23. YOSHIDA, C. F. T.; NOGUEIRA, R. M. R.; MERCADANTE, L. A. C; PINHÃO, A. T.; SCHATZMAYR, H. G. Seroepidemiological survey of Hepatitis A and B, Cytomegalovirus and Herpes simplex type 2 in prime blood donors from Rio de Janeiro, Brazil. Rev. Microbiol. S. Paulo, 18: 5-11, 1987. Recebido para publicação em 29/8/1991 Reapresentado em 22/11/1991 Aprovado para publicação em 6/12/1991
https://openalex.org/W2191834086	https://nottingham-repository.worktribe.com/preview/770107/JJennings-BCP-CO2-manuscript_revised.pdf	English	null	How does dense phase CO<sub>2</sub> influence the phase behaviour of block copolymers synthesised by dispersion polymerisation?	Polymer chemistry	2,016	cc-by	9,780	How does dense phase CO2 influence the phase behaviour of block copolymers synthesised by dispersion polymerisation? J. Jennings,a S. P. Bassett,a D. Hermida-Merino,b G. Portale, b W. Bras,b L. Knight,a J. J. Titman,a T. Higuchi,c H. Jinnai, c S. M. Howdle a a School of Chemistry, University of Nottingham, University Park, Nottingham, NG7 2RD, UK. E-mail: steve.howdle@nottingham.ac.uk; Fax: +0044 115 846 8459; Tel: +44 (0)115 9513486 bDUBBLE@ESRF, Netherlands Organisation for Scientific Research (N.W.O.), CS40220, 38043, Grenoble, Cedex 9, France c Institute of Multidisciplinary Research for Advanced Materials (IMRAM), Tohoku University, 2-1-1 Katahira, Aoba-ku, Sendai 980-8577, Japan a School of Chemistry, University of Nottingham, University Park, Nottingham, NG7 2RD, UK. E-mail: steve.howdle@nottingham.ac.uk; Fax: +0044 115 846 8459; Tel: +44 (0)115 9513486 Abstract Block copolymers synthesised in supercritical CO2 dispersion undergo in situ self-assembly which can result in a range of nanostructured microparticles. However, our previous study revealed that copolymers with different block combinations possessed different microphase separated morphologies at identical block volume fractions. In this paper, we follow up those initial observations. By examining the phase behaviour of a selection of structurally diverse block copolymers, we explore the structural factors which influence the conflicting self- assembly behaviours. The composition dependence of the morphology is found to be strongly related to the CO2-philicity of the second block relative to poly(methyl methacrylate) (PMMA). Whilst PMMA-b-poly(benzyl methacrylate) (PBzMA) and PMMA-b-poly(N,N- dimethylaminoethylmethacrylate) (PDMAEMA) phase behaviour follows traditional diblock copolymer phase diagrams, PMMA-b-poly(styrene) (PS) and PMMA-b-poly(4-vinyl pyridine) (P4VP), which comprise blocks with the greatest contrast in CO2-philicity, self- assemble into unexpected morphologies at several different block volume fractions. The morphology of these copolymers in the microparticulate form was found to revert to the predicted equilibrium morphology when the microparticles were re-cast as films and thermally annealed. These findings provide strong evidence that CO2 acts as a block-selective solvent during synthesis. The CO2-selectivity was exploited to fabricate various kinetically trapped non-lamellar morphologies in symmetrical PMMA-b-PS copolymers by tuning the ratio of polymer:CO2. Our data demonstrate that CO2 can be exploited as a facile process modification to control the self-assembly of block copolymers within particles. Introduction Block copolymer self-assembly is an important phenomenon that has facilitated the development of new areas of research and applications in material science.1 The driving force for polymer-polymer phase separation is the enthalpic incompatibility of two or more chemically distinct polymers. In block copolymers, the presence of covalent bonds between the polymers prevents macrophase separation and results instead in microphase separated domains typically on the order of 10-100 nm in size. To achieve self-assembly in diblock copolymers, the two blocks must have a sufficiently high Flory-Huggins interaction parameter (χ), and degree of polymerisation (N). Through theoretical and experimental studies, it has been elucidated that the product N must exceed a critical value of ~10.5 in order for a diblock copolymer to overcome the entropic penalty of chain stretching and microphase separate.2 The appearance of the morphology depends on the relative block volume fraction (f) of the two blocks, the most common being lamellar, bicontinuous (e.g. double gyroid3 or double diamond4), hexagonally-packed cylinders and body centered cubic spherical phase, listed in order of deviation from flat interfacial curvature. These diverse structures and the length-scales at which they exist have been exploited for many nanotechnology applications including nanocomposite synthesis,5 bottom up lithography6 and photonic crystals.7 Confinement of self-assembled block copolymers in three dimensions (i.e. in nano or microparticles, nanorods etc.) has further expanded the field and potential scope of applications. In addition to conventional morphologies, new frustrated structures have been observed in block copolymer nanoparticles including mushroom, screw-like and helical morphologies which occur when the size of the confining particle approaches the range of block domain sizes.8, 9 Block copolymer particles have inspired investigations into a range of functional materials for use in drug delivery,10, 11 synthesis of mesostructured inorganic materials12, bio-imaging13 and metamaterials.14 A number of methods exist for the fabrication of microphase separated block copolymer particles. The most common techniques to date are those which exploit solvent evaporation- induced self-assembly. For example, the self-organised reprecipitation (SORP) method involves slow solvent evaporation from block copolymer dissolved in a solvent/non-solvent mixture. This results in block copolymer particles formed by precipitation, in which self- assembly can be induced either by means of solvent15 or thermal annealing.16 Other related methods exploit solvent evaporation from block copolymers dissolved in dispersed particles17 or aerosols.11, 18 However, the drawbacks of these methods include the requirement for multiple steps (i.e. Block copolymer structural characterisation The block copolymer synthetic procedure is outlined in the supporting information. Block copolymers were analysed by 1H NMR in CDCl3 on a Bruker DPX 300 MHz spectrometer in order to determine the mass fraction of the blocks. This was converted to volume fraction, fPMMA, using the melt densities, where available (PMMA (1.17 g cm-3), PBzMA (1.179 g cm- 3), PS (1.05 g cm-3) and P4VP (1.15 g cm-3)).25 Molecular weight (Mn,exp) and dispersity (Đ) were determined by GPC using either an Agilent PL GPC 120 in THF or a PL GPC 50 in a mixture of chloroform/ethanol/triethylamine (90/10/0.5 by volume). Analyses were run at a flow rate of 1 mL min-1 and 40 °C, and columns were calibrated with PMMA narrow standards. Introduction block copolymer synthesis followed by solvent evaporation and annealing) and the use of volatile organic solvents. An alternative route is the use of emulsion, miniemulsion or dispersion polymerisations in which block copolymers are synthesised and self-assembly occurs within the particle during polymerisation. These techniques typically exploit controlled radical polymerisation (CRP) techniques in green solvents including water19-21 and supercritical carbon dioxide (scCO2).22, 23 Solvophobic polymerisations provide an attractive route to block copolymer microparticles since they are relatively facile, green and industrially applicable with fewer processing steps. However, both green solvents have drawbacks: water consumes significant energy in drying and waste water must be cleaned up after use; and scCO2 requires specialised high pressure equipment.24 Recently we reported a route to block copolymers which takes advantage of the excellent livingness of RAFT polymerisation in a dispersion polymerisation in scCO2. This proved to be a particularly effective method to access a range of block copolymer particles with a wide array of nanostructured morphologies.22, 23 Understanding the factors governing the phase behaviour of block copolymer particles synthesised in scCO2 is crucial for establishing structure-property relationships for the design of new materials for novel applications. Herein we investigate the possible influence of CO2 on the morphology of several block copolymers synthesised by dispersion polymerisation in the medium. The equilibrium structure of the block copolymers was studied by preparing solution cast films, and further insight into the effect on the morphology by CO2 was gained by modulating the polymer:CO2 ratio through adjustment of monomer loading. The results provide insight into the mechanism of formation, and possible new methods by which to exert control of block copolymer morphology within microparticles. Experimental Block copolymer structural characterisation Transmission electron microscopy (TEM) Block copolymer particles were embedded in epoxy resin (Agar 100) and set at 35 °C for 72 h before being ultramicrotomed at room temperature to ~100 nm slices with a diamond knife (Leica Diatome Ultra 45°) and collected on copper grids. Sections of PMMA-b-PBzMA were stained with RuO4 for ∼1 h, which adsorbs selectively to PBzMA domains. PMMA-b-PSt particles were stained prior to resin embedding with OsO4 for 24 h, which adsorbs selectively to PS. Sections of PMMA-b-P4VP and PMMA-b-PDMAEMA were stained with I2 vapour for ∼2 h, which selectively adsorbs to P4VP and PDMAEMA domains. Imaging of particle samples took place on either a JEOL 200FXII or a FEI Tecnai microscope. Block copolymer films were prepared by solvent casting. PMMA-b-PS and PMMA-b- PBzMA particles were dissolved in toluene at 1 wt% and cast as films before being annealed at 160 °C in vacuo for 2 days. PMMA-b-P4VP particles were dissolved in chloroform at 2 wt% and cast as films, before being annealed in a saturated atmosphere of chloroform vapour. The films were then embedded in an acrylic resin and ultramicrotomed at room temperature with a diamond knife. Sections were collected on copper grids and stained with RuO4 (PMMA-b-PBzMA and PMMA-b-PS) or I2 (PMMA-b-P4VP) for 8 h. TEM imaging of the films was conducted at 200 kV. Small Angle X-ray Scattering (SAXS) SAXS data were collected at the ESRF (Grenoble) at a sample-to-detector distance of either ~3 m or 6.150 m with a wavelength of 1.033 or 0.8266 Å, respectively. A Dectris-Pilatus 1M detector with a resolution of 981 x 1043 pixels and a pixel size of 172 x 172 μm was employed to record the 2D scattering profiles. Standard corrections for sample absorption and background subtraction were performed. The data were normalised with respect to the incident beam intensity in order to correct for primary beam intensity fluctuations. The scattering patterns from rat tail collagen or silver behenate were used for the calibration of the wave vector scale of the scattering curve. Bulk films or neat block copolymer particles were placed in the beamline and SAXS patterns were acquired at room temperature. The scattering vector q is defined as q = 4π/λ sin θ, where 2θ is the scattering angle. Domain spacing, D, was calculated from D = 2π/qmax, where qmax is the position of the principle scattering peak. Solid State NMR (SS NMR) SS NMR data were acquired on a Bruker Avance III spectrometer operating at 600.13 MHz using a 1.3 mm HX MAS probe with a spinning rate of 60 kHz. A simple three-pulse exchange pulse sequence was used to acquire two-dimensional 1H-1H spin diffusion spectra. The spectral width in both dimensions was set to 50 ppm. To obtain a complete diffusion profile between 13-17 spectra were acquired for each sample using mixing times of duration 1 ms to 700 ms. Pure phase lineshapes were obtained using TPPI, and a z-alternation phase cycle was used, as described by Spiess and Schmidt-Rohr.26 The relaxation delay was between 5 and 7 s depending on the sample, so that the total acquisition time for a single 2D 1H-1H spin diffusion spectrum was around 3 hours. Deconvolution of the spectra was carried out by fitting 16 two-dimensional peaks to the 2D data surface using mixed Lorentzian/Gaussian peak shapes with fixed positions and varying widths and amplitudes. Intensities were obtained by taking the volume integrals of the fitted peaks. These were corrected for T1 relaxation and then normalised against the total magnetisation present at short mixing times. The change intensity of the polystyrene aromatic diagonal peak showed two linearly decaying regions corresponding to intra-domain and inter- domain spin diffusion. The second shallower decay was extrapolated to the time taken for full equilibration of the magnetisation and this time was then used to calculate a domain size according to the methods described by Spiess and co-workers.26, 27 T1 values were recorded for all samples using separate saturation-recovery experiments. Simple one-dimensional spectra were recorded to obtain the equilibrium magnetisation levels. To obtain an independent measure of the spin diffusion coefficients in the two polymer domains, average distances between different 1H sites in a single repeating unit were calculated using QChem.28 The average difference between the PS aromatic and the aliphatic hydrogens was 0.50 nm and the average distance between the PMMA methoxy and methyl hydrogens was 0.56 nm. Results and Discussion s of PMMA-containing block copolymers were synthesised in scCO2 dispersion.22, 23 A series of PMMA-containing block copolymers were synthesised in scCO2 dispersion.22, 23 Efficient RAFT control over MMA polymerisation was demonstrated, with PMMA dispersity in the range 1.2-1.5. Chain extension to block copolymer was observed by the increase in molecular weight by GPC, while dispersity remained relatively low (mostly <1.7). As previously, higher dispersities were recorded for some PMMA-b-PS and PMMA-b-P4VP copolymers, where termination during the second block polymerisation occurred by combination and lead to a high molecular weight shoulder in the GPC trace (SI Figure 1). Second blocks were selected that differed in CO2-philicity: from the more CO2-philic poly(benzyl methacrylate) (PBzMA) and poly(N,N-dimethylaminoethyl methacrylate) (PDMAEMA), to the more CO2-phobic polystyrene (PS) and poly(4-vinylpyridine) (P4VP). Block copolymer particles with a range of block volume fractions and molecular weights were synthesised via dispersion polymerisation in scCO2 to understand the effect of scCO2 on the final particle internal morphology. A comparison of the morphology within particles obtained directly from scCO2 and after thermal annealing was then carried out to probe the persistence of the obtained structure. Furthermore, the equilibrium structure of the block copolymers was studied by preparing solution cast films and correlating the morphologies obtained with literature examples of these block copolymers synthesised by standard methods. Finally, to gain further insight into the effect on the morphology by CO2, the polymer to CO2 ratio was varied by synthesising block copolymers at different monomer loadings. Block copolymers with various volume fractions were synthesised targeting two molecular weights by a RAFT-controlled dispersion polymerisation and morphology investigated through TEM analysis (Table 1). Polymers are named according to the theoretical number average molecular weights of the two blocks in the copolymer, e.g. PMMAx-P2y, where x and y represent the target molecular weight of PMMA and block P2 in the copolymer (in kg mol- 1), calculated from the molar ratio of monomer to RAFT agent used in the synthesis. average molecular weights of the two blocks in the copolymer, e.g. PMMAx-P2y, where x and y represent the target molecular weight of PMMA and block P2 in the copolymer (in kg mol- 1), calculated from the molar ratio of monomer to RAFT agent used in the synthesis. average molecular weights of the two blocks in the copolymer, e.g. Results and Discussion PMMAx-P2y, where x and y represent the target molecular weight of PMMA and block P2 in the copolymer (in kg mol- 1), calculated from the molar ratio of monomer to RAFT agent used in the synthesis. Table 1: Characteristics of block copolymers synthesised by RAFT dispersion in scCO2 for phase behaviour studies Block copolymer name Mn,exp a (/103 g mol-1) Đa fPMMA b Morphologyc PMMA22.5- PBzMA37.5 54 1.60 0.39 LAM PMMA30-PBzMA30 57 1.53 0.51 LAM/DIS PMMA37.5-PBzMA22.5 55 1.24 0.61 LAM/CYL PMMA45-PBzMA15 51 1.35 0.72 SPH/DIS PMMA37.5-PBzMA62.5 73 1.26 0.38 LAM PMMA50-PBzMA50 77 1.38 0.51 LAM PMMA62.5-PBzMA37.5 73 1.66 0.64 LAM/BIC PMMA75-PBzMA25 70 1.45 0.78 SPH PMMA30-PDMAEMA30 44 1.33 0.58 LAM PMMA45-PDMAEMA15 48 1.24 0.83 CYL PMMA22.5-PS37.5 55 1.42 0.36 LAM PMMA30-PS30 54 1.69 0.48 CYL PMMA37.5-PS22.5 46 1.67 0.61 SPH PMMA45-PS15 49 1.81 0.76 SPH PMMA37.5-PS62.5 71 1.57 0.38 LAM PMMA50-PS50 73 1.83 0.48 SPH PMMA62.5-PS37.5 68 1.93 0.61 SPH PMMA75-PS25 71 1.97 0.77 SPH PMMA15-P4VP45 61 1.71 0.67 LAM PMMA30-P4VP30 68 1.98 0.49 SPH PMMA45-P4VP15 69 1.99 0.29 SPH aDerived from GPC analysis in THF (PMMA-b-PBzMA and PMMA-b-PS) or CHCl3/EtOH/TEA (PMMA-b-PDMAEMA and PMMA-b-P4VP) analysed against PMMA standards. bCalculated from the weight fraction of PMMA derived from 1H NMR analysis. cDetermined by TEM imaging: LAM (lamellar), BIC (bicontinuous), CYL (cylindrical), SPH (spherical) and DIS (disordered morphology) or combinations thereof racteristics of block copolymers synthesised by RAFT dispersion in scCO2 haviour studies All-methacrylic block copolymers PMMA-b-PBzMA copolymer particles synthesised with different volume fractions in scCO2 displayed a multitude of morphologies (Figure 1), ranging from lamellar to bicontinuous, cylindrical and spherical (in the latter two PMMA comprises the matrix), as the final volume fraction of PMMA in the copolymer increased. TEM imaging revealed evidence of the coexistence between nanostructured and disordered particles within certain PMMA-b- PMMA-b-PBzMA copolymer particles synthesised with different volume fractions in scCO2 displayed a multitude of morphologies (Figure 1), ranging from lamellar to bicontinuous, cylindrical and spherical (in the latter two PMMA comprises the matrix), as the final volume fraction of PMMA in the copolymer increased. TEM imaging revealed evidence of the coexistence between nanostructured and disordered particles within certain PMMA-b- PBzMA copolymer samples (SI Figure 2). SAXS analysis was conducted in order to investigate whether the majority of particles within the sample existed in the nanostructured or disordered state (SI Figure 3). Results and Discussion The absence of any Bragg scattering in PMMA30-PBzMA30 and PMMA45-PBzMA15, leads us to believe that these samples consisted of mostly disordered particles (as notified in Table 1), signifying that PMMA-b-PBzMA was in the weak segregation limit and blocks are highly miscible at this molecular weight. The only other literature in which PMMA-b-PBzMA was studied by TEM found that a copolymer of ~15 kg mol-1 (synthesised in a heterogeneous polymerisation) was miscible and also formed disordered block copolymer particles.29 Figure 1: TEM images of cross-sectioned PMMA-b-PBzMA particles prepared at different final volume fractions and molecular weights, displaying a variety of morphologies: Lamellar (LAM), bicontinuous (BIC), lamellar/cylindrical coexistence (LAM/CYL) and spherical/disorder coexistence (SPH/DIS). The scale bar in all images is 500 nm. LAM BIC LAM/CYL SPH/DIS PMMA50-PBzMA50 PMMA62.5-PBzMA37.5 PMMA37.5-PBzMA22.5 PMMA45-PBzMA15 LAM BIC LAM/CYL SPH/DIS PMMA50-PBzMA50 PMMA62.5-PBzMA37.5 PMMA37.5-PBzMA22.5 PMMA45-PBzMA15 LAM PMMA50-PBzMA50 BIC PMMA62.5-PBzMA37.5 PMMA50-PBzMA50 PMMA62.5-PBzMA37.5 PMMA62.5-PBzMA37.5 Figure 1: TEM images of cross-sectioned PMMA-b-PBzMA particles prepared at different final volume fractions and molecular weights, displaying a variety of morphologies: Lamellar (LAM), bicontinuous (BIC), lamellar/cylindrical coexistence (LAM/CYL) and spherical/disorder coexistence (SPH/DIS). The scale bar in all images is 500 nm. The order and final volume fraction at which the different phases formed in PMMA-b- PDMAEMA was consistent with those in PMMA-b-PBzMA. The symmetrical copolymer self-assembled into lamellar morphology, whilst cylindrical morphology (in which PMMA comprised the matrix) formed at a higher PMMA block volume fraction (Figure 2). self-assembled into lamellar morphology, whilst cylindrical morphology (in which PMMA comprised the matrix) formed at a higher PMMA block volume fraction (Figure 2). Figure 2: TEM images of cross-sectioned PMMA-b-PDMAEMA particles prepared at different final volume fractions and molecular weights, displaying a variety of morphologies: Lamellar (LAM) and cylindrical (CYL). The scale bar in both images is 200 nm. LAM A CYL PMMA30-PDMAEMA30 PMMA45-PDMAEMA15 A CYL PMMA45-PDMAEMA15 LAM PMMA30-PDMAEMA30 PMMA30-PDMAEMA30 Figure 2: TEM images of cross-sectioned PMMA-b-PDMAEMA particles prepared at different final volume fractions and molecular weights, displaying a variety of morphologies: Lamellar (LAM) and cylindrical (CYL). The scale bar in both images is 200 nm. The morphologies observed by TEM for PMMA-b-PBzMA were plotted in the form of an experimental phase diagram (Figure 3). The appearance of self-assembled morphologies at their respective block volume fractions suggested that PMMA-b-PBzMA (and PMMA-b- PDMAEMA) adhered to traditional behaviour of linear diblock copolymers. Thus, it appeared that these block copolymers synthesised in dispersion polymerisation in scCO2 were minimally perturbed from equilibrium. Results and Discussion Figure 3: Experimental phase diagram for PMMA-b-PBzMA synthesised in scCO2, constructed based on TEM images of block copolymers in Table 1. Block copolymers observed in this study (■) are plotted as a function of their relative degree of polymerisation (no χ interaction parameter data were available for this polymer pair) against calculated block volume fraction. Morphologies are abbreviated to LAM (lamellar), BIC (bicontinuous), CYL (cylindrical), SPH (spherical). χN (arbitrary) fPMMA χN (arbitrary) fPMMA Figure 3: Experimental phase diagram for PMMA-b-PBzMA synthesised in scCO2, constructed based on TEM images of block copolymers in Table 1. Block copolymers observed in this study (■) are plotted as a function of their relative degree of polymerisation (no χ interaction parameter data were available for this polymer pair) against calculated block volume fraction. Morphologies are abbreviated to LAM (lamellar), BIC (bicontinuous), CYL (cylindrical), SPH (spherical). Figure 3: Experimental phase diagram for PMMA-b-PBzMA synthesised in scCO2, constructed based on TEM images of block copolymers in Table 1. Block copolymers observed in this study (■) are plotted as a function of their relative degree of polymerisation (no χ interaction parameter data were available for this polymer pair) against calculated block volume fraction. Morphologies are abbreviated to LAM (lamellar), BIC (bicontinuous), CYL (cylindrical), SPH (spherical). Methacrylic-styrenic block copolymers The phase behaviour of PMMA-b-PS has been well studied in the literature in thin films and bulk,30, 31 and under spherical confinement within microparticles formed via SORP.32 Thus, this system provides an ideal opportunity to elucidate any effects that are specific to block copolymer structures produced in scCO2. In particular, the higher CO2-philicity of PMMA over PS is well-founded.33 Characterisation by TEM revealed a range of morphologies from lamellar to cylindrical and spherical (in which PMMA comprised the matrix) as PMMA block volume fraction increased (Figure 4). Unlike PMMA-b-PBzMA, only one copolymer (PMMA45-PS15) showed coexistence of nanostructured and disordered particles, and the appearance of a strong Bragg peak in SAXS analysis suggested that the sample comprised nanostructured particles as the majority (SI Figure 4). Figure 4: TEM images of cross-sectioned PMMA-b-PS particles prepared at different final volume fractions and molecular weights, displaying a variety of morphologies: Lamellar (LAM), cylindrical (CYL) and spherical (SPH). The scale bar in all images is 200 nm. PMMA22.5-PS37.5 LAM CYL SPH SPH PMMA30-PS30 PMMA50-PS50 PMMA45-PS15 PMMA22.5-PS37.5 LAM PMMA22.5-PS37.5 SPH PMMA50-PS50 SPH PMMA45-PS15 SPH Figure 4: TEM images of cross-sectioned PMMA-b-PS particles prepared at different final volume fractions and molecular weights, displaying a variety of morphologies: Lamellar (LAM), cylindrical (CYL) and spherical (SPH). The scale bar in all images is 200 nm. The experimental phase diagram (Figure 5) clearly revealed that the phase behaviour for PMMA-b-PS deviated from PMMA-b-PBzMA and PMMA-b-PDMAEMA, and from PMMA-b-PS reported previously. In our study, the symmetrical PMMA50-b-PS50 (fPMMA = 0.48) with total molecular weight of 73 kg mol-1 showed spherical morphology. Lamellar morphology has been widely observed in films and particles of PMMA-b-PS at comparable molecular weights and volume fractions30, 32, 34 χN fPMMA Figure 5: Experimental phase diagram for PMMA-b-PS synthesised in scCO2, constructed based on TEM images of block copolymers in Table 1. Block copolymers observed in this study (■) are plotted as a function of χN using literature values of χ for PMMA-b-PS at 65 °C.35 Morphologies are abbreviated to L (lamellar), C (cylindrical) and S (spherical). The drastically different appearance to the phase diagram of PMMA-b-PBzMA strongly suggests that CO2 is affecting the phase behaviour of these structurally distinct copolymers. TEM imaging of PMMA-b-P4VP (Figure 6) revealed phase behaviour more consistent with PMMA-b-PS than PMMA-b-PBzMA or PMMA-b-PDMAEMA. In particular, the symmetrical copolymer (fPMMA = 0.49) showed spherical morphology (in which PMMA formed the matrix), whilst the more P4VP-rich copolymer (fPMMA = 0.29) showed lamellae. TEM imaging of PMMA-b-P4VP (Figure 6) revealed phase behaviour more consistent with PMMA-b-PS than PMMA-b-PBzMA or PMMA-b-PDMAEMA. In particular, the symmetrical copolymer (fPMMA = 0.49) showed spherical morphology (in which PMMA formed the matrix), whilst the more P4VP-rich copolymer (fPMMA = 0.29) showed lamellae. Figure 6: TEM images of cross-sectioned PMMA-b-P4VP particles prepared at different final volume fractions, displaying a variety of morphologies: spherical (SPH) and lamellar (LAM). The scale bar in all images is 200 nm. SPH LAM PMMA30-P4VP30 PMMA15-P4VP45 SPH PMMA30-P4VP30 SPH Figure 6: TEM images of cross-sectioned PMMA-b-P4VP particles prepared at different final volume fractions, displaying a variety of morphologies: spherical (SPH) and lamellar (LAM). The scale bar in all images is 200 nm. Block copolymer annealing Block copolymer annealing Block copolymer annealing Comparison of phase behaviour of all methacrylic vs. methacrylic-styrenic block copolymers confirmed that block volume fraction is not the only factor influencing phase behaviour of block copolymers synthesised in scCO2 dispersion. In particular, the morphologies formed in methacrylic-styrenic copolymers were more curved away from PMMA at the same volume fractions. The methacrylic-styrenic copolymers differed from all methacrylics in that they displayed higher dispersities, and also higher homopolymer contamination, as estimated by chromatography in our previous report.23 Block dispersity has been observed to influence diblock copolymer self-assembly, resulting in morphologies with increased curvature toward the more polydisperse domain.36 In addition to this, homopolymer contamination could lead to discrepancies in block volume fraction which could also explain the deviations in phase behaviour for the methacrylic-styrenic copolymers. fPMMA Thus, to investigate whether structural variables caused the deviation in block copolymer phase behaviour, solvent cast bulk films were prepared from the scCO2 synthesised particles to effect a more thermodynamically stable state of the various block copolymers (Figure 7). Figure 7: TEM images of block copolymer bulk films prepared by directly solvent casting and annealing the scCO2 synthesised microparticles. Scale bar in each image is 200 nm. CYL LAM LAM PMMA50-PBzMA50 PMMA30-PS30 PMMA15-P4VP45 CYL PMMA15-P4VP45 LAM PMMA30-PS30 LAM PMMA50-PBzMA50 Figure 7: TEM images of block copolymer bulk films prepared by directly solvent casting and annealing the scCO2 synthesised microparticles. Scale bar in each image is 200 nm. In the symmetrical PMMA50-PBzMA50, the bulk film displayed lamellar morphology, consistent with the as-synthesised particles (Figure 1). However, the symmetrical block copolymer of PMMA30-PS30, which displayed cylindrical morphology in microparticles (Figure 4), showed lamellar morphology in the film. Finally, PMMA15-P4VP45, which had lamellar morphology in the particles (Figure 6), showed a disordered cylindrical morphology, In the symmetrical PMMA50-PBzMA50, the bulk film displayed lamellar morphology, consistent with the as-synthesised particles (Figure 1). However, the symmetrical block copolymer of PMMA30-PS30, which displayed cylindrical morphology in microparticles (Figure 4), showed lamellar morphology in the film. Finally, PMMA15-P4VP45, which had lamellar morphology in the particles (Figure 6), showed a disordered cylindrical morphology, in which P4VP was the matrix, in the bulk film form. Due to the slightly ambiguous nature of the TEM image of PMMA15-P4VP45 film, SAXS analysis was also performed in order to confirm the assignment (SI Figure 5). Overall, the observed bulk film morphologies were consistent with traditional block copolymer self-assembly. The morphology of the symmetrical PMMA-b-PS in particles has also been found to be lamellar, which rules out the possibility of spherical confinement influencing the phase behaviour.32 Frustration arising from spherical confinement is typically observed when the ratio of particle diameter to domain spacing is smaller than 2,8 a ratio which we far exceed in our present study. Clearly, the morphologies that we obtained in the microparticles directly from scCO2 are kinetically trapped, and thermodynamically-stable morphologies are only obtained after these particles have been dissolved and then solvent cast into bulk films. This strongly indicates that the contrast in CO2-philicity was influencing phase behaviour in these block copolymer systems. fPMMA Although scCO2 is the non-solvent for dispersion polymerisation, it is absorbed by many polymers, resulting in a swelling and plasticisation.37 Thus, it likely has some influence on the dispersed particles of block copolymer synthesised in situ. Studies into block copolymer phase behaviour in the presence of CO2 yield often conflicting results. In some cases, the miscibility of the two blocks is increased, as unfavourable enthalpic interactions are screened by CO2 sorption.38 In block copolymers with lower disorder-order transitions (LDOT), CO2 sorption can lower the temperature at which phase separation occurs by increasing compressibility of polymer chains, which entropically disfavours mixing.39 Finally, order- order transitions (OOTs) can be affected when the superior solubility of CO2 in a CO2-philic block (e.g. fluorinated or siloxane polymers) results in an increase in effective block volume fraction and hence a phase transition40-42. We speculated that the polymer structure-dependent solubility of CO2 influenced the phase behaviour of methacrylic-styrenic block copolymers relative to all methacrylics in the manner of a block selective solvent during the synthesis.22 Domain size measurements In addition to SAXS and TEM measurements, solid-state NMR spin diffusion experiments were carried out to obtain an additional measure of the average domain size throughout the whole sample. Ultrafast magic angle spinning (MAS) 1H – 1H two-dimensional exchange spectra were recorded with increasing mixing times43, and the changes in peak intensities were used to monitor the transfer of magnetization between the domains by spin diffusion. This approach can be applied here, because the two domains are rigid, so that the spin-lattice relaxation times are relatively long. Figure 8 shows the resulting decay in the intensity of the diagonal peak in the two-dimensional spectrum corresponding to the polystyrene aromatic 1H sites for three samples: PMMA37.5-PS22.5, PMMA62.5-PS37.5 and PMMA50-PS50. The initial steep decay results from the re-equilibration of magnetization between side-group aromatic and backbone aliphatic sites within the PS blocks, while the subsequent shallower decay occurs as magnetization is transferred to the PMMA domains. For the latter the time taken for the magnetisation to decay to its equilibrium value is related to the domain size of the polymer which can be calculated using the method previously described by Speiss.26, 27 The resulting domain sizes are compared with those obtained from SAXS and TEM in Table 2. Further experimental details are given in the Supplementary Information). To further probe the kinetically-trapped nature of the morphologies observed in PMMA-b-PS particles, samples were thermally annealed above the glass transition temperature (Tg), but below the order-disorder transition temperature. This processing imparted polymer mobility to facilitate the return to the preferred block copolymer morphology in the absence of CO2. A number of PMMA-b-PS samples were analysed by SAXS before and after thermal annealing experiments. The position of Bragg reflections at similar q values before and after thermal treatment suggested that no major morphological shifts (i.e. order-order transitions) took place, although this could not be confirmed due to the lack of higher order peaks. This implied that microphase separation remained localised within particles, and no long range order developed during the annealing experiment. Interestingly, an increase in domain size was registered after thermal treatment for PMMA-b- PS particles at all volume fractions and molecular weights (measured by SAXS only). Since the domain spacing in a block copolymer is proportional to χN, the smaller domain size in the as-synthesised particles could be a result of the decrease in χ by CO2. Domain size measurements A decrease in χ for block copolymers annealed in CO2 has been previously reported, and can be explained by block miscibilisation.38 Thus, CO2 was most likely decreasing χ between PMMA and PS, affecting smaller domain sizes, which further hinted that a kinetically-trapped morphology synthesised in scCO2 was returning to the thermodynamically favoured state over the course of the annealing experiment. Table 2: PMMA-b-PS domain size measurements before and after thermal annealing Table 2: PMMA-b-PS domain size measurements before and after thermal annealing aDetermined by TEM imaging: LAM (lamellar), CYL (cylindrical), SPH (spherical). bCalculated from solid state NMR 1H-1H spin diffusion spectra; cDerived from the principal scattering peak (qmax) in the SAXS profile using D = 2π/qmax Block Copolymer Morphologya DTEM a (nm) DNMR b (nm) DSAXS c (nm) DSAXS an neal c (nm) PMMA22.5-PS37.5 LAM 33 24 37 40 PMMA30-PS30 CYL 35 38 39 n/a PMMA37.5-PS22.5 SPH 41 33 39 40 PMMA45-PS15 SPH 44 30 25 29 PMMA37.5-PS62.5 LAM 48 50 54 59 PMMA50-PS50 SPH 76 60 62 n/a PMMA62.5-PS37.5 SPH 55 41 49 53 PMMA75-PS25 SPH 55 41 46 48 Figure 8. Decay of the diagonal peak corresponding to aromatic PS 1H sites from ultrafast MAS 1H - 1H two-dimensional exchange NMR spectra as a function of mixing time τm for three copolymers: PMMA37.5-PS22.5 (filled circles) PMMA62.5-PS37.5 (grey squares) and PMMA50-PS50 (open circles). The lines show linear extrapolations used to find the mixing time when re-equilibration is achieved, from which the domain size can be extracted. Figure 8. Decay of the diagonal peak corresponding to aromatic PS 1H sites from ultrafast MAS 1H - 1H two-dimensional exchange NMR spectra as a function of mixing time τm for three copolymers: PMMA37.5-PS22.5 (filled circles) PMMA62.5-PS37.5 (grey squares) and PMMA50-PS50 (open circles). The lines show linear extrapolations used to find the mixing time when re-equilibration is achieved, from which the domain size can be extracted. Block copolymers synthesis at variable monomer loading p y y g In the presence of a selective solvent, block copolymer self-assembly is influenced by the volume fraction of block copolymer relative to solvent.44 Increasing the concentration of a selective solvent enhances swelling and therefore effective volume fraction of one block, resulting in morphologies with increasing curvature away from the block for which the solvent is selective. By synthesising block copolymers at different monomer loading, the volume fraction of the final copolymer in CO2, Φcopolymer, could be adjusted. Symmetrical PMMA-b-PBzMA and PMMA-b-PS copolymers were synthesised at two monomer loadings, targeting two molecular weights, and the phase behaviour studied by TEM (Table 3, Figures 9 and 10). Table 2: PMMA-b-PS domain size measurements before and after thermal annealing Table 3: Characteristics of block copolymers synthesised by RAFT dispersion in scCO2 at various monomer loadings Block copolymer name Mn,exp a (/103 g mol-1) Đa Φcopolymer b Morphologyc PMMA30- PBzMA30-16ml 54 1.60 0.225 LAM/DIS PMMA30-PBzMA30-25ml 57 1.53 0.347 LAM/DIS PMMA50-PBzMA50-16ml 77 1.38 0.225 LAM PMMA50-PBzMA50-25ml 92 1.24 0.347 LAM PMMA30-PS30-16ml 55 1.42 0.237 CYL PMMA30-PS30-25ml 54 1.69 0.367 LAM PMMA50-PS50-16ml 77 1.86 0.237 SPH PMMA50-PS50-25ml 91 1.48 0.367 CYL aDerived from GPC analysis in THF (PMMA-b-PBzMA and PMMA-b-PS) or analysed against PMMA standards. bCalculated from Φcopolymer = (νMMA + νmonomer-2) / (𝑣𝐶𝑂2 + νMMA + νmonomer-2) cDetermined by TEM imaging: LAM (lamellar), CYL (cylindrical), SPH (spherical) and DIS (disordered) Table 3: Characteristics of block copolymers synthesised by RAFT dispersion in scCO2 at various monomer loadings against PMMA standards. bCalculated from Φcopolymer = (νMMA + νmonomer-2) / (𝑣𝐶𝑂2 + νMMA + νmonomer-2) cDetermined by TEM imaging: LAM (lamellar), CYL (cylindrical), SPH (spherical) and DIS (disordered) For PMMA-b-PBzMA, the expected lamellar morphology was observed at both molecular weights and regardless of Φcopolymer (Figure 9). For a block copolymer in a neutral solvent, morphology should remain constant with Φcopolymer.45 This suggests an equal swelling of the structurally similar methacrylate blocks by CO2 and no influence on relative block volume fractions, as expected. fractions, as expected. Figure 9: TEM images of cross-sectioned particles of PMMA30-PBzMA30 (top) and PMMA50- PBzMA50 (bottom) synthesised at two polymer volume fractions in CO2 (Φcopolymer). Scale bar in all images is 500 nm. Morphology was evidently independent of the polymer concentration in CO2. PMMA30- PBzMA30-16ml PMMA30-PBzMA30-25ml PMMA50-PBzMA50-16ml PMMA50-PBzMA50-25ml LAM/DIS LAM/DIS LAM LAM PMMA30- PBzMA30-16ml PMMA30-PBzMA30-25ml PMMA50-PBzMA50-16ml PMMA50-PBzMA50-25ml LAM/DIS LAM/DIS LAM LAM PMMA30- PBzMA30-16ml PMMA50-PBzMA50-16ml LAM Figure 9: TEM images of cross-sectioned particles of PMMA30-PBzMA30 (top) and PMMA50- PBzMA50 (bottom) synthesised at two polymer volume fractions in CO2 (Φcopolymer). Scale bar in all images is 500 nm. Morphology was evidently independent of the polymer concentration in CO2. On the contrary, particles synthesised under the same conditions for PMMA-b-PS showed a polymer volume fraction-dependent morphology at both molecular weights (Figure 10). Specifically, as Φcopolymer increased, a transition from cylindrical to lamellar morphology occurred for PMMA30-PS30 (Figure 10, top) and spherical to cylindrical for PMMA50-PS50 (Figure 10, bottom). Figure 10: TEM images of cross-sectioned particles of PMMA30-PS30 (top) and PMMA50 – PS50 (bottom) synthesised at two polymer volume fractions in CO2 (Φcopolymer). Scale bar in all images is 500 nm. Table 2: PMMA-b-PS domain size measurements before and after thermal annealing Morphology was heavily influenced by polymer concentration in CO2. PMMA50-PS50-16ml PMMA50-PS50-25ml PMMA30-PS30-16ml PMMA30-PS30-25ml CYL LAM SPH CYL CYL PMMA50-PS50-16ml SPH PMMA50-PS50-25ml CYL CYL Figure 10: TEM images of cross-sectioned particles of PMMA30-PS30 (top) and PMMA50 – PS50 (bottom) synthesised at two polymer volume fractions in CO2 (Φcopolymer). Scale bar in all images is 500 nm. Morphology was heavily influenced by polymer concentration in CO2. As Φcopolymer was decreased (i.e. CO2 concentration increased), the morphology shifted across the phase diagram towards phases which were more highly curved away from PMMA. This is consistent with selective swelling of PMMA. The fact that CO2 is selective for methacrylates over styrenics is well known, and numerous reports have quantified a greater degree of CO2 absorbed and thus volume increase in such polymers.33 This is thought to be a result of Lewis base-Lewis acid interactions between the carbonyl oxygen lone pair and the electropositive carbon in CO2.46 This degree of relative volume increase can be controlled by varying the polymer volume fraction in CO2, which materialises in a shift in self-assembled morphology. This is likely also the cause of the shifted phase behaviour in the structurally related PMMA- b-P4VP copolymer system, The fact that the non-equilibrium morphology is maintained on removal of CO2 strongly suggests a trapping mechanism. At some critical point during the polymerisation, when molecular weight exceeds the critical χN (which will be influenced by the presence of CO2 and monomer), microphase separation takes place. We have developed a high pressure cell for future in situ monitoring of polymerisation and elucidation of the phase separation onset by SAXS.47 Once all monomer is consumed, the final block copolymer morphology will be governed by the block volume fraction of CO2-swollen PMMA relative to the 2nd block. On cooling the reaction, CO2 remains between the polymer chains and morphology is arrested as both blocks pass below their Tg (lowered by CO2 plasticisation) and chains cannot reorganise. Once CO2 is removed from the polymerisation reactor, the morphology is unable to return to the thermodynamically favoured state based on polymer block volume fraction alone. Thus, the resulting morphology is kinetically-trapped and the CO2 selective solvent effect persists. Conclusions We report detailed studies of the self-assembly of a number of block copolymers synthesised by RAFT-controlled dispersion polymerisation in scCO2. Experimental phase diagrams for methacrylic-styrenic block copolymer systems differed from all-methacrylic systems in terms of the self-assembled morphologies at a range of block volume fractions. These data suggested that structural ambiguities result in differential absorption of CO2 which leads to the formation of kinetically-trapped morphologies. This effect was confirmed by preparing bulk films under thermodynamic control, in which morphologies returned to those predicted by volume fraction, and the observed increase in domain size upon thermal annealing. Furthermore, by varying the ratio of polymer to CO2, a morphological influence could be imparted by a selective solvent effect, which hints at an entirely new route to control nanostructured morphology of block copolymer particles in an already industrially-amenable synthetic process. The ability to create a range of morphologies from a single copolymer composition could be exploited for applications such as drug delivery vehicles, impact modifiers or even light scattering / light absorbing materials, in which properties will be dependent on internal nanostructured morphology. 4. Chu, C. Y.; Jiang, X.; Jinnai, H.; Pei, R. Y.; Lin, W. F.; Tsai, J. C.; Chen, H. L., Real- space evidence of the equilibrium ordered bicontinuous double diamond structure of a diblock copolymer. Soft Matter 2015, 11 (10), 1871-1876. Table 2: PMMA-b-PS domain size measurements before and after thermal annealing Others have shown that RAFT dispersion polymerisation in aqueous or alcoholic media can be used to create a range of self-assembled structures (worms, vesicles, etc.)48 but there are no published examples of microparticulate structures, as highlighted earlier.22 Others have demonstrated formation of particles by heterogeneous CRP, but by use of emulsion and miniemulsion rather than dispersion, and all of these studies clearly produce only particles with lamellar morphologies at symmetric volume fractions19, 49, or less well-defined morphologies that are trapped by crosslinking,21 again demonstrating that the scCO2 dispersion route is unusual. Previous studies have found that non-equilibrium morphologies can be frozen into films of PS-b-PFOMA,40 PS-b-PFMA41, 50 and PDMS-b-PMPCS42 after annealing in CO2 (PFOMA, PFMA and PDMS are highly CO2-philic blocks). Significantly, our data appear to be the first examples of CO2 behaving as a block-selective solvent in block copolymers comprising only highly CO2-phobic blocks. This effect might be enhanced by the confinement of block copolymer to microparticles rather than thin films which are typically studied. In thin films, there is the additional variable of polymer-substrate interaction. This can result in CO2 concentration enrichment at the substrate relative to the polymer film,51 which may reduce the influence of CO2 on the block copolymer self-assembly. The ability to tune morphology as a function of polymer concentration in CO2 hints at an alternative and facile method of influencing phase separated morphology of industrially relevant block copolymer particles. Supporting Information Block copolymer synthetic procedure, additional TEM images of block copolymer particles, raw SAXS data from block copolymer particles and films, SS NMR. 3. Jinnai, H.; Nishikawa, Y.; Spontak, R. 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https://openalex.org/W4285013954	https://journal.uinsgd.ac.id/index.php/publica/article/download/17764/pdf	English	null	The Effectiveness of SP4N-LAPOR-Based Complaints! At the Department of Communication and Information Technology of Tasikmalaya City	Publica/Publica	2,022	cc-by-sa	5,439	Abstract When the public service process is still not maximized, the community will submit a complaint report to the Government on the suggested complaints from the community regarding public services that have not been effective. The presence of SP4N-LAPOR! is a need for a system that can integrate all complaints management in government agencies, because complaints cannot be managed partially. The purpose of this study was to determine the effectiveness of SP4N-LAPOR! at the Department of Communication and Information of the City of Tasikmalaya. This study uses the theory of Sedarmayanti (2009) regarding the effectiveness of the program with 4 indicators, namely: Input, Production Process, Output (Output), Productivity. Using descriptive research methods with a qualitative approach. Data collection techniques by observation, interviews, and documentation. Data analysis used Miles and Huberman (1984) in Sugiyono (2019), namely data collection, data reduction, data presentation and conclusion drawing/verification. The result of this research is that the complaints based on SP4N-LAPOR! has been effective in accordance with the existing mechanism in the SOP, but there are still obstacles, namely limited human resources and employee understanding and not optimal in conducting socialization. Keywords: Local Government, E-Goverment, Program Effectivity, SP4N-LAPOR! 1Universitas Muhammadiyah Jakarta, Indonesia; fitri.gupitasari24@gmail.com 1Universitas Muhammadiyah Jakarta, Indonesia; fitri.gupitasari24@gmail.com Received: January 7, 2022; In Revised: May 10, 2022; Accepted: June 13, 2022 Fitri Gupitasari, Muhammad Khoirul Anwar The Effectiveness of SP4N-LAPOR-Based Complaints! In Tasikmalaya City Communication and Information Office Publica: Jurnal Pemikiran Administrasi Negara Vol 14 No. 1 \| Juni 2022: 1-10 Publica: Jurnal Pemikiran Administrasi Negara Vol 14 No. 1 \| Juni 2022: 1-10 P-ISSN 2085-6555 E-ISSN 2715-9256 P-ISSN 2085-6555 E-ISSN 2715-9256 The Effectiveness of SP4N-LAPOR-Based Complaints! At the Department of Communication and Information Technology of Tasikmalaya City P-ISSN 2085-6555 E-ISSN 2715-9256 addressed to the community which is the special handling of complaints. In line with Law No. 25 of 2009 on Public Services, employees are required to provide the best service and facilitate access to the community while the aim is to convey messages to employees in providing their services. The People's Online Aspirations and Complaints Service System (LAPOR!) is an online- based complaint facility. Established and managed by the Presidential Work Unit for Development Supervision and Control (UKP4). Based on Presidential Regulation Number 76 of 2013 and Minister of State Apparatus Empowerment and Bureaucratic Reform Regulation Number 3 of 2015, LAPOR! designated as the National Public Service Complaint Management System (SP4N). REPORT! used to involve community participation and improve two-way relations between the people and the government in supervising development programs. SP4N-REPORT! now connected with 34 Ministries, 96 Institutions, and 493 Local Governments in Indonesia. When a report is received through the SP4N-LAPOR! can categorize according to the categories available on the system. The following features are included in SP4N-LAPOR! First of all, the tracking id is a unique number, which functions to monitor the process of following up on aspiration reports from the community. The second is anonymous, which is a feature to hide identity data when making reports. The identity of the reporting party will not be known or read by the reported party or the general public. And the third secret is a report that comes in but the report will not be seen by the general public. In Tasikmalaya City Mayor Regulation Number 37 of 2019 concerning Guidelines for Managing Public Complaints About Public Services in Tasikmalaya City, the Government provides for the general public in terms of expressing the complaint stage. When the community is not dissatisfied with government services at the regional level, the community has the right to submit reports, either verbally or in writing on paper. Reports submitted by part of the community to the government which are part of the public service, are accepted by the community for the government. Any complaints that come in, especially regarding the implementation and development of public services will be submitted to the relevant parties within the Tasikmalaya city officials for further processing. Then on the theory of effectiveness that effectiveness comes from "effective" which means that it achieves and gets success by achieving the process of the goals that have been set. Fitri Gupitasari, Muhammad Khoirul Anwar The Effectiveness of SP4N-LAPOR-Based Complaints! In Tasikmalaya City Communication and Information Office Publica: Jurnal Pemikiran Administrasi Negara Vol 14 No. 1 \| Juni 2022: 1-10 Publica: Jurnal Pemikiran Administrasi Negara Vol 14 No. 1 \| Juni 2022: 1-10 P-ISSN 2085-6555 E-ISSN 2715-9256 P-ISSN 2085-6555 E-ISSN 2715-9256 Introduction In the digital age, the Government is always trying to transform. With the development of communication and information technology, it can provide solutions in the performance of public services based on good governance. E-government provides opportunities to improve the quality of government through increasing efficiency, providing new services, and increasing public participation. The use of technology can simplify the way the bureaucracy works and make it more innovative. Good innovation can provide convenience for bureaucrats and the public as beneficiaries of public service providers. With the existence of this system, the work of the bureaucracy can finally respond competitively and give advantages to the state. Based on the Presidential Instruction of the Republic of Indonesia Number 3 of 2003 concerning the National Policy and Strategy for the Development of e-Government that the Government must carry out the demands of the community which includes the affairs of the general public by upholding accountability, being actively accessible and listening to the aspirations of the community as well as providing communication facilities in formulating a policy. One of the conveniences for the public to submit suggestions, complaints and aspirations is through complaints, where the handling of these complaints is the responsibility of the government. As stated in Article 36 and Article 37, handling complaints is the duty and responsibility of the government. As stated in the law that regulates public services, it is 1 P-ISSN 2085-6555 E-ISSN 2715-9256 This effectiveness has to do with the relationship between the results obtained. Effective service obtains various points of view (view points) and gets an assessment by having links and various ways that are very closely related to efficiency. The Liang Gie (Halim, 2004), says the opinion about effectiveness which sees the existence of a situation that does occur to the will of a person by treating the act with a certain and desired purpose, then the person is said to be effective, if the result arises or has the intended purpose. that person. According to (Siagian, 2018) Effectiveness is the facilities and infrastructure and resources with a certain amount that has been determined to produce a number of activities that have been carried out. With the effectiveness that can show the success of the actions that have been set goals. The more these activities lead to action from the goals/targets, the higher the effectiveness of the activities. Effectiveness is that there are main elements in order to achieve the goals or objectives that have been previously completed in activities, programs or organizations. According to Mahmudi in the journal (Fatmala, 2019) expressed an opinion about the effectiveness that is part of the relationship between goals and output, with an increase in the contribution of the output regarding the acquisition of goals, that it increases the effectiveness of activities, a set 2 Publica: Jurnal Pemikiran Administrasi Negara Vol 14 No. 1 \| Juni 2022: 1-10 P-ISSN 2085-6555 E-ISSN 2715-9256 P-ISSN 2085-6555 E-ISSN 2715-9256 P-ISSN 2085-6555 E-ISSN 2715-9256 of programs or organizations. Based on the view that the existence of an effectiveness has a reciprocal interaction between goals and outputs. According to Sedarmayanti (2009), expressed his opinion, namely an effectiveness that embodies a form of measurement that continues the description of the concept of the targets it has achieved. In this way, the effectiveness manifests itself in more measures that continue to the concept of targets that have been previously set by the organization to achieve what is achieved. Some of the definitions of effectiveness that are part of the experts can be concluded that the effectiveness of improving welfare for the community through certain programs, according to him, is very important for the community to have very important goals with a better development process. P-ISSN 2085-6555 E-ISSN 2715-9256 E-government (Indrajit, 2005), is an effort to create an atmosphere of government service that is in line with the shared objectives (Shared Goals) of a number of interested communities, therefore the vision made must reflect the shared vision of stakeholders, such as improving productivity and government operational performance related to public services; Promote governance with integrity and transparency; Improving the quality of life of the community with the performance of community services; and Ensure the creation of a democratic state administration. Backus (2001) expressed an opinion related to E-government regarding the form of e- business implemented by the government, which refers to structures and processes aimed at providing public services in electronic or digital form aimed at entrepreneurs and the general citizens. Meanwhile, according to Sudrajat et al (2015) in the journal of Wulandari (2019) E- government is an information technology process in disseminating and collecting information for a government purpose. According to the World Bank (2000) in (Habibullah, 2010), with the implementation of E-government to facilitate transactions as well as progress for the utilization of technology for banking around the world. Growing e-government is aimed at advancing the level of efficiency, accountability, and transparency of government management by using digital technology or other internet. Meanwhile, according to the Government of Indonesia, E-government is an application of information technology, internet-based and other digital devices, which are run by the government for the purpose of delivering information from the government to the public, business partners, employees, business entities, and other institutions online. According to Moenir (2006) in the book Mulyawan (2016), service is a fulfillment for the needs of community activities with the aim of taking care of or preparing what is needed for others. Public service is defined as public or community. According to Syafiie (2010) an understanding of the public which consists of several social beings who have togetherness to express feelings, with ways of thinking, attitudes, hopes and the existence of good and right actions, on the basis of their norms and values. Thus, public service is an activity that is often carried out to help the general public. Concept of the National Public Service Complaint Management System (SP4N) The implementation of the management of the national public complaint system is the integration of complaint management in stages at each provider within the framework of the public service information system (PERPRES Number 76/2013). Fitri Gupitasari, Muhammad Khoirul Anwar The Effectiveness of SP4N-LAPOR-Based Complaints! In Tasikmalaya City Communication and Information Office Fitri Gupitasari, Muhammad Khoirul Anwar Publica: Jurnal Pemikiran Administrasi Negara Vol 14 No. 1 \| Juni 2022: 1-10 P-ISSN 2085-6555 E-ISSN 2715-9256 P-ISSN 2085-6555 E-ISSN 2715-9256 P-ISSN 2085-6555 E-ISSN 2715-9256 The Memorandum of Understanding was then followed up in the form of a Cooperation Agreement. Based on the Cooperation Agreement between the 3 institutions, the responsibilities of these three institutions are in implementing SP4N and using the LAPOR! can be broken down as follows: Kemenpan RB as the coordinator of SP4N is responsible for preparing the policy framework, socializing the implementation, providing guidance related to the implementation of the mechanism, managing complaint reports through the LAPOR system! SP4N and supervise the management performance. The Presidential Staff Office (KSP) as the provider of access and technical support for the LAPOR!-SP4N system is in charge of managing the LAPOR! SP4N, provides support in the utilization of the LAPOR!-SP4N application, maintains and develops LAPOR!-SP4N application tools and systems, provides support and facilities for complaint application integration, provides guidance modules and organizes LAPOR! system training, and uses LAPOR! data. to carry out the function of monitoring and controlling development. The Ombudsman of the Republic of Indonesia as the supervisor of the SP4N implementation is responsible for managing and resolving complaints through the LAPOR! SP4N especially in the case of delays in the completion of the handling of complaints, managing reports with complaints that are disposed of to the Ombudsman of the Republic of Indonesia from the LAPOR! system, and taking the necessary follow-up actions to encourage changes to a data-based system. From this explanation, the researcher estimates that the effectiveness of the SP4N- LAPOR! the Tasikmalaya City Communication and Information Office can influence the smooth running of the tasks/functions of the Tasikmalaya City Government in an effort to improve public services, as well as knowing the extent of the SP4N-LAPOR! effective. Fitri Gupitasari, Muhammad Khoirul Anwar The Effectiveness of SP4N-LAPOR-Based Complaints! In Tasikmalaya City Communication and Information Office P-ISSN 2085-6555 E-ISSN 2715-9256 The National Public Service Complaint Management System (SP4N) is an integrated system in the management of complaints in stages at each organizer within the framework of a public service information system (Permenpan No. 24/2014). 3 Publica: Jurnal Pemikiran Administrasi Negara Vol 14 No. 1 \| Juni 2022: 1-10 Jabatan For data validation, Triangulation technique is used as a technique to review the validity of the data. Triangulation can be done using different techniques (Nasution, 2003) namely interviews, observations and documents. This triangulation is not only used to review the validity of the data but also to enrich the data. According to Nasution, besides that triangulation can also be useful to investigate the validity of the researcher's interpretation of the data, because triangulation is reflective. Miles and Huberman (1984) in (Sugiyono, 2019) conducted qualitative data analysis to be carried out interactively and continuously until it was completed. Activities in data analysis, namely data reduction data, presenting data, and drawing conclusions. In this study, the data obtained in the form of a description of the results of the interview process. The data that has been obtained is then processed and interpreted so that researchers can find and understand the implied meaning of the state of the subject. Process and analyze data systematically so that later the data obtained is of high quality. The location for conducting research is at the Department of Communication and Information of the City of Tasikmalaya, Jalan Ir. H. Juanda No. 191, Sukamulya Village, Bungursari District, Tasikmalaya City, West Java 46151. This research was carried out with initial observations on 27 September 2021 and for in-depth research carried out in January - March 2022. Fitri Gupitasari, Muhammad Khoirul Anwar The Effectiveness of SP4N-LAPOR-Based Complaints! In Tasikmalaya City Communication and Information Office Publica: Jurnal Pemikiran Administrasi Negara Vol 14 No. 1 \| Juni 2022: 1-10 P-ISSN 2085-6555 E-ISSN 2715-9256 P-ISSN 2085-6555 E-ISSN 2715-9256 P-ISSN 2085-6555 E-ISSN 2715-9256 According to Moleong (2017) explaining an informant must have a lot of experience related to the research background. The criteria for an informant are mastering the problem, having data, and being willing to convey accurate information. Informants were determined using the purposive sampling technique, which is the process of determining informants based on certain criteria. Informants in this study are: Methods The type of research approach used is a qualitative approach with a descriptive method. The qualitative approach according to Sugiyono (2019), which has the principle of a research work mechanism, which is guided by non-statistical or non-mathematical subjective assessments. The measure of value in qualitative research is not score numbers, but the categorization of quality values. While the descriptive method according to Sugiyono (2019), by describing the results carefully will later be in the form of painting, depicting, or exposing the condition of the object being studied as it is, according to the situation and conditions when the research was conducted. According to Sugiyono (2019), data collection techniques are the most important step in research, because the main purpose of research is to obtain data, including through observations or observations made through observations and recordings to obtain more accurate information about the things being studied. related to the process in the SP4N-LAPOR! at the Department of Communication and Information of the City of Tasikmalaya. Then interviews were conducted to collect materials or information, based on the questions and answers process orally and face to face with research informants, carried out in depth to be considered to understand the problems studied. The interview chosen by the researcher was an unstructured interview. And on the documentation technique at the time of taking pictures or data needed by the author at the Office of Communication and Information Technology of the City of Tasikmalaya, in the form of soft copies of data and photos related to the implementation of services. 4 Publica: Jurnal Pemikiran Administrasi Negara Vol 14 No. 1 \| Juni 2022: 1-10 Fitri Gupitasari, Muhammad Khoirul Anwar Table 1 Table 1 Research Informants Nama Jabatan Informan B Sub Coordinator for Information, Communication, and Public Informan A JFT Skilled Computer Administrator and SP4N-LAPOR Admin Informan R JFT Skilled Computer Administrator and SP4N-LAPOR Admin Source: Processed by Authors, 2022 Results and Discussion The determination of the change in the status of a Village to a Village is stated in Regional Regulation No. 30 of 2003 dated October 31, 2003. Through this regional regulation, the City of Tasikmalaya has an area of 69 Kelurahan. Based on Regional Regulation No. 6 of 2008 concerning the formation of Bungursari and Purbaratu sub-districts, which are divisions of Indihiang and Cibeureum sub-districts, thus the number of sub-districts in the Tasikmalaya city area becomes 10 sub-districts, including: Bungursari sub-district, Cibeureum sub-district, Cihideung sub-district, Cipedes sub-district, Indihiang sub-district, sub-district Kawalu, Mangkubumi District, Purbaratu District, Tamansari District, Tawang District 5 Publica: Jurnal Pemikiran Administrasi Negara P-ISSN 2085-6555 Vol 14 No. 1 \| Juni 2022: 1-10 E-ISSN 2715-9256 =========================================================================== Image 1 Total Population and Population Density by District in 2022 Source: data.tasikmalayakota.go.id Publica: Jurnal Pemikiran Administrasi Negara Vol 14 No. 1 \| Juni 2022: 1-10 P-ISSN 2085-6555 E-ISSN 2715-9256 P-ISSN 2085-6555 E-ISSN 2715-9256 Total Population and Population Density by District in 2022 Total Population and Population Density by District in 2022 Total Population and Population Density by District in 2022 Source: data.tasikmalayakota.go.id Source: data.tasikmalayakota.go.id The results of this study are based on the results of data and facts in the field based on the results of interviews with informants who refer to indicators of program effectiveness (Sedarmayanti, 2009). These indicators are used as a measure of how the effectiveness of the program has been implemented or not. So that the results of this study can be used as material for consideration by the agency in order to achieve the goals to be achieved. The results of this study are described as follows: Fitri Gupitasari, Muhammad Khoirul Anwar The Effectiveness of SP4N-LAPOR-Based Complaints! In Tasikmalaya City Communication and Information Office Fitri Gupitasari, Muhammad Khoirul Anwar The Effecti eness of SP4N LAPOR Based Compl Publica: Jurnal Pemikiran Administrasi Negara Vol 14 No. 1 \| Juni 2022: 1-10 Publica: Jurnal Pemikiran Administrasi Negara Vol 14 No. 1 \| Juni 2022: 1-10 P-ISSN 2085-6555 E-ISSN 2715-9256 P-ISSN 2085-6555 E-ISSN 2715-9256 Fitri Gupitasari, Muhammad Khoirul Anwar The Effectiveness of SP4N-LAPOR-Based Complaints! In Tasikmalaya City Communication and Information Office 2. Production Process The production process is a system that plays a role in processing inputs to realize the planned goals, based on the results achieved. In order for this production process to be carried out successfully, by means of communication and socialization, it will lead to a good interaction between the community and the Government, in addition to decision making and development of the apparatus, with the existence of these points in order to find out how clear the time must be in a plan and can improve the performance of the apparatus. On the SP4N-REPORT! This will run more optimally when in planning, do not forget to focus on a budget, where the budget will be prepared within one year. Regarding the source of funds or budget, this is in accordance with the Mayor's Regulation in article 20, namely, the financing for handling public complaints is charged to the Tasikmalaya City Regional Revenue and Expenditure Budget. The funding issued by the Tasikmalaya City Discominfo is the cost for making leaflets and posters when the apparatus socializes the SP4N-LAPOR-based public complaint program! to society. In this socialization and communication, the SP4N-LAPOR! at Diskominfo Tasikmalaya City provide understanding to the public regarding how to report complaints and convey aspirations. 1. Whistleblower: For the public in submitting a complaint or aspiration report to SP4N- LAPOR! can go through the website lapor.go.id, SMS 1708, and can download the SP4N- LAPOR! application. Reports must be verified first by the report admin! for clarity and completeness and for further disposition by certain agencies. 1. Whistleblower: For the public in submitting a complaint or aspiration report to SP4N- LAPOR! can go through the website lapor.go.id, SMS 1708, and can download the SP4N- LAPOR! application. Reports must be verified first by the report admin! for clarity and completeness and for further disposition by certain agencies. 2. Follow-up report: The relevant agency will give time according to the classification of the report, when the complaint report does not have a level of supervision, it is 14 days, while for a report with a level of supervision it is more than 14 days. However, the SOP only takes 5 working days to carry out internal coordination and prepare follow-up reports provided by the community. Furthermore, the agency will confirm through the comment column. 3. 1. Input Input is data or information needed by the system for further processing in accordance with predetermined specifications. Input is the initial stage of implementing the objectives which will then enter the system. Each input has a purpose of facilities and infrastructure, human resources, and available funding sources for improvement. In the process of implementing the vision and mission through planning, good input is needed so that the goals and targets can be carried out as planned, so public complaints to the Government are based online using SP4N-LAPOR!. There are 34 employees at the Tasikmalaya City Diskominfo, and the SP4N-LAPOR admin manager! totaling 3 people. The Tasikmalaya City Diskominfo apparatus is tasked with conducting socialization and being the admin coordinator of the City Government with the aim of distributing or disposing of reports of complaints or aspirations that are submitted to the City Government. if the report enters the realm of government, the Diskominfo will submit it to the central government, but if it is for other regional governments, it will be sent to several regional governments directly. As for the supporting facilities used by Diskominfo, it only requires a computer and internet network, each SP4N-LAPOR! At Diskominfo, it is given to hold 1 computer unit in managing complaints and aspirations of the community so that they can be assigned to the related OPD. On the SP4N-REPORT! There are features that are beneficial to society. Such as the anonymous feature where the identity of the reporter will not be known by the public, the Government and others, the secret feature makes the report invisible to the public and the tracking id feature is a unique number to review the follow-up process for reports submitted by the complainant. 6 P-ISSN 2085-6555 E-ISSN 2715-9256 2. Production Process End of report: The report is completed when the relevant agency or service has followed up on the report and if within 10 working days after the follow-up there is no response, the report will be considered complete and the report will be deemed to have come from the reporting party. 3. End of report: The report is completed when the relevant agency or service has followed up on the report and if within 10 working days after the follow-up there is no response, the report will be considered complete and the report will be deemed to have come from the reporting party. Furthermore, in making decisions on how to receive public complaints through the admin, report to the Diskominfo of Tasikmalaya City. From the point of view of Diskominfo, if a complaint comes in, the admin coordinator of SP4N-LAPOR! The Diskominfo of Tasikmalaya City immediately made a disposition, while the resolution of the problem was by a certain party because it was not the authority of the Diskominfo, but for incomplete complaints such as no names, locations not listed, and incomplete chronology, the admin coordinator of SP4N-LAPOR! at Diskominfo did not follow up on it so it had to be postponed before being archived. In the sense that it is returned to the complainant that the report is not complete and must be completed if it is to be followed up. 7 Publica: Jurnal Pemikiran Administrasi Negara P-ISSN 2085-6555 Vol 14 No. 1 \| Juni 2022: 1-10 E-ISSN 2715-9256 =========================================================================== Image 2 Number of Incoming Complaint Reports Source: lapor.go.id, 2021 Publica: Jurnal Pemikiran Administrasi Negara P-ISSN 2085-6555 Vol 14 No. 1 \| Juni 2022: 1-10 E-ISSN 2715-9256 =========================================================================== Image 2 Number of Incoming Complaint Reports Source: lapor.go.id, 2021 Publica: Jurnal Pemikiran Administrasi Negara Vol 14 No. 1 \| Juni 2022: 1-10 P-ISSN 2085-6555 E-ISSN 2715-9256 Number of Incoming Complaint Reports Source: lapor.go.id, 2021 Source: lapor.go.id, 2021 p , The Effectiveness of SP4N-LAPOR-Based Complaints! In Tasikmalaya City Communication and Information Offi Fitri Gupitasari, Muhammad Khoirul Anwar 3. Hasil/Output Effectiveness can be seen from a person's performance. Personal performance is how employees do their jobs. Therefore, improving employee performance greatly affects the performance of the organization where the employee is involved in achieving predetermined organizational goals. In addition, job satisfaction in the workplace can be used as input, assuming that job satisfaction is a condition that can indicate a person's performance. So that the results (Output) given by the Diskominfo of Tasikmalaya City are in the form of success in providing services related to disposition, and filtering in managing reports of complaints and public aspirations. The following data is seen from the large number of reports submitted in the last 1 year, there are 64 reports that the ability of the SP4N-LAPOR admin apparatus! In completing the process of reporting complaints and public aspirations, the Diskominfo of Tasikmalaya City has been completed or has been assigned to the relevant OPD, but for November and December 2021 it is still in the process stage. Image 3 Monthly Incoming Report Details Source: lapor.go.id, 2021 Fitri Gupitasari, Muhammad Khoirul Anwar The Effectiveness of SP4N-LAPOR-Based Complaints! In Tasikmalaya City Communication and Information Office 8 Image 3 Monthly Incoming Report Details Source: lapor.go.id, 2021 Monthly Incoming Report Details Fitri Gupitasari, Muhammad Khoirul Anwar 8 Fitri Gupitasari, Muhammad Khoirul Anwar The Effectiveness of SP4N-LAPOR-Based Complaints! In Tasikmalaya City Communication and Information Office Fitri Gupitasari, Muhammad Khoirul Anwar The Effectiveness of SP4N-LAPOR-Based Complaints! In Tasikmalaya City Communication and Information Office Publica: Jurnal Pemikiran Administrasi Negara Vol 14 No. 1 \| Juni 2022: 1-10 P-ISSN 2085-6555 E-ISSN 2715-9256 P-ISSN 2085-6555 E-ISSN 2715-9256 P-ISSN 2085-6555 E-ISSN 2715-9256 When a report comes in from Permenpan to Diskominfo, it is necessary to verify first, including where the report is, because for now there is a new system in which the report goes to the level of supervision or not to the level of supervision, then directly distributed to the OPD, and from the OPD a maximum of 5 -7 days for the complaint has been responded to by the agency. If the report has been followed up, then there is a point for the apparatus, namely regarding rewards where the community can give a rating for the services provided by the Communication and Information Office to the community regarding the management of complaints reports and community aspirations. 3. Hasil/Output As can be seen, the Tasikmalaya City Diskominfo is the admin coordinator for SP4N- LAPOR! to manage the report, the maximum of the existing SOPs is 3 days, sometimes when the incoming report is complete, it is immediately disposed of. In addition, it can be seen from how effective it is in resolving complaints using the SP4N-LAPOR! it's been effective. Not only that, in terms of communication between the two directions, from the community and government perspective, it has been effective. So that the Diskominfo of Tasikmalaya City is ranked 2nd in West Java. 4. Productivity Productivity in Sedarmayanti's book education is related to how to produce quality graduates, according to needs. By improving the quality of education, it is hoped that graduates will be able to become employees who can carry out their job duties better. So that in the application of public complaints based on SP4N-LAPOR! This requires quality human resources, this educational component will be a measuring tool in the implementation of SP4N- LAPOR! especially in the scope of Diskominfo Tasikmalaya City. To see the work productivity of a graduate depends on the input given at the time when they are given training or technical guidance to manage SP4N-LAPOR!. When the admin of the Tasikmalaya City Diskominfo coordinator is quick to respond or work according to the SOPs applied in managing reports of complaints or aspirations of the people that come in. Education for SP4N-LAPOR Admins carried out by the Kemenpan RB in Jakarta for 3 years related to the management of SP4N-LAPOR!. In educational supplies SP4N-LAPOR!, this is done every time there are changes or additions to features related to the lapor.go.id website. The Tasikmalaya City Diskominfo also provides information door to door visiting SKPD and provides technical guidance to SP4N-LAPOR admins! OPD. This will continue and be maintained because the information and knowledge gained through this educational provision will not stop at one person, but everyone can know this knowledge. Fitri Gupitasari, Muhammad Khoirul Anwar The Effectiveness of SP4N-LAPOR-Based Complaints! In Tasikmalaya City Communication and Information Office Conclusion The community will submit a complaint report to the Government on complaints suggested by the community regarding ineffective public services. One of the conveniences for the public to express their aspirations and complaints is through SP4N-LAPOR!. As for the results of observations, interviews and documentation on the SP4N-LAPOR-based public complaints program that the input has not been effective due to limited human resources, especially in the related OPD, thus affecting the handling of complaints. In HR planning, it is better to focus on certain steps taken by management in order to ensure that an organization provides the right workforce. In the production process, it is quite effective in disposing of reports because it is in accordance with the target or SOP. Furthermore, the results/outputs have been effective, in the implementation of managing SP4N-LAPOR! seen from the performance of employees in managing incoming reports so as to get satisfaction from the community, by showing good performance can increase work productivity. The last point in terms of 9 Publica: Jurnal Pemikiran Administrasi Negara Vol 14 No. 1 \| Juni 2022: 1-10 P-ISSN 2085-6555 E-ISSN 2715-9256 P-ISSN 2085-6555 E-ISSN 2715-9256 productivity has been effective because when this is presented, the Government provides first briefing and training to admins on how to manage public complaints, so that this training and debriefing will affect the success of a program planning. Fitri Gupitasari, Muhammad Khoirul Anwar p , The Effectiveness of SP4N-LAPOR-Based Complaints! In Tasikmalaya City Communication and Information Offi Fitri Gupitasari, Muhammad Khoirul Anwar The Effectiveness of SP4N-LAPOR-Based Complaints! In Tasikmalaya City Communication and Information Office References Backus, M. (2001). E-Governance and Developing. IICD Research Brief. Fatmala, R., Kahar, F. (2019). Efektivitas Program Pelayanan Badan Penyelenggara Jaminan Sosial (Bpjs) Kesehatan Di Puskesmas Libureng Kabupaten Bone. Birokrat : Jurnal Ilmu Administrasi Publik. 8 (2). Habibullah, A. (2010). Kajian Pemanfaatan dan Pengembangan E-government. Ilmu Administrasi Negara, FISIP, Universitas Jember. 23 (3). hlm. 187-195. Halim, A. (2004). Pengelolaan Keuangan Daerah. Yogyakarta: UPP STIM YKPN. Indrajit, R. E. (2005). e-Government In Action. Malang: Andi Offset. Moleong. (2017). Metodologi Penelitian Kualitatif. Jakarta Timur: PT. Remaja Rosdakary Mulyawan, R. (2016). Birokrasi dan Pelayanan Publik. Bandung: UNPAD PRESS. Nasution, S. (2003). Metode Research (Penelitian Ilmiah). Jakarta: Bumi Aksara. Pasolong, M. (2019). Teori Administrasi Publik. Jakarta: CV. Alfabeta Peraturan Walikota Tasikmalaya Nomor 37 Tahun 2019 Tentang Pedoman Pengelolaan Pengaduan Masyarakat Tentang Pelayanan Publik Di Kota Tasikmalaya. Peraturan Walikota Tasikmalaya Nomor 87 Tahun 2020 Tentang Tugas Pokok Dan Rincian Tugas Unit Dinas Komunikasi Dan Informatika Kota Tasikmalaya. Peraturan WaliKota Tasikmalaya Nomor 55 Tahun 2020 Tentang Susunan Organisasi, Kedudukan, Tugas Pokok, Fungsi Dan Tata Kerja Perangkat Daerah. Panduan Untuk Penyelenggara Dan Administrator pada Pemerintah Provinsi/Kabupaten/Kota 2016. Peraturan Menteri Pendayagunaan Aparatur Negara Dan Reformasi Birokrasi Republik Indonesia Nomor 62 Tahun 2018 Tentang Pedoman Sistem Pengaduan Pelayanan Publik Nasional. Sedarmayanti, M. A. (2009). Sumber Daya Manusia dan Produktivitas Kerja. Bandung: Mandar Maju. Siagian, S. P. (2018). Manajemen Sumber Daya Manusia. Jakarta: Bumi Aksara. Sugiyono. (2019). 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https://openalex.org/W4379520460	https://www.degruyter.com/document/doi/10.1515/9783111241463-005/pdf	English	null	The Study of Religion and the Spirit of Orientalism: Cultural Transformations and Scholarly Shifts	De Gruyter eBooks	2,023	cc-by	8,696	1 This text was read (on zoom) as a keynote lecture at the Conference of the European Academy of Religion in Münster on September 1, 2021. I wish to thank Professor Hans-Peter Grosshans for his kind invitation to deliver the lecture. The text is adapted from the introduction to my Guy G. Stroumsa, The Idea of Semitic Monotheism. The Rise and Fall of a Scholarly Myth. Oxford: Oxford University Press 2021. Open Access. © 2023 Guy G. Stroumsa, published by De Gruyter. This work is licensed under the Creative Commons Attribution 4.0 International License. https://doi.org/10.1515/9783111241463-005 Guy G. Stroumsa The Study of Religion and the Spirit of Orientalism: Cultural Transformations and Scholarly Shifts Like all social institutions, religions constantly change, evolving and transforming themselves. Sometimes, when one can speak of brutal transformations and of reli- gious revolutions, this truism is obvious to all.1 But more often, the conatus of reli- gions entails their more or less ‘peaceful’ evolution. Now, while all religions have a potential for radical transformation in their DNA, they are instinctively more inclined toward conservatism, and the function of their hermeneutical systems, while permitting change when deemed necessary or unavoidable, consists essen- tially in slowing down the pace of such change, and making sure, it goes unnoticed, or almost unnoticed. Daniel Kahneman, a psychologist who won a Nobel Prize in economics, has shown that we all function with two parallel systems of thought: ‘Thinking, Fast and Slow,’ as he entitled his book on the topic. Similarly, I propose we identify two alternate patterns of religious change: slow versus fast. Religion and society at large, then, are complementary motors, actors, and objects of change in history. In a permanent dialectical relationship with one another, religion and society are taking turns at initiating change, responding to it, modulating it, and transforming the direction, intensity, and even the very nature of this change. In a recently published book, The Idea of Semitic Monotheism: The Rise and Fall of a Scholarly Myth, I sought to understand some societal dimensions of the idea of Semitic monotheism, an idea coined by Ernest Renan in the mid-19th century and developed in European scholarship in the subsequent decades. This idea was interdependent with both new perceptions of Judaism and Islam and transformed attitudes toward Jews and Muslims. Hence, I tried to highlight various veiled con- nections between the history of religions, then a growing scholarly discipline, and some deep changes in European societies, related to secularization processes. I shall present a few preliminary reflections on this research here, which, I think, bear directly on the topic of our conference, from a particular but capital angle. It barely needs mentioning that since the early modern Great Discoveries 64 Guy G. Stroumsa Guy G. Stroumsa 64 and especially since the industrial revolution, the pace of change has been faster than ever before. Guy G. Stroumsa The Study of Religion and the Spirit of Orientalism: Cultural Transformations and Scholarly Shifts The contemporary entry into the Digital Age has transformed the world, indeed is still transforming it in many ways, into what we have learned to call a ‘global village’, although a thoroughly polluted one, far from any idyllic or Romantic imagination. With the Enlightenment, in particular, Europe, and then many societies all around the world, have undergone a complex process of height- ened secularization – a process jump-started by the Reformation. While seculari- zation is a highly polyvalent term, it always refers to a shrinking of the status of established or organized religion in the public sphere, including personal law, the limitation of its political role, and the enhanced focus of the private and personal sphere as the main or legitimate locus of religion. It is clear, then, that the process of secularization has a deep impact on the nature of religion, involving far-reaching changes first in its practice but also in its doctrine as well as in its self-perception. With those changes in religion, another, perhaps subtler but no less significant phenomenon was initiated by the critique of religion at the time of the Enlighten- ment. The non-theological approach of Christianity, and by extension of Judaism and Islam, the other monotheistic religions best known in Europe, also permitted the growth of the modern study of religion, and modeled anew the approach to religious phenomena. I wish to argue that a major paradigm shift in the very per- ception of monotheism, and in particular of Judaism and Islam, occurred through- out the 19th century, and that this paradigm shift bears directly on the formation of modern scholarship on religion. Towards the end of the 19th century, the ‘science of religion’ stood (unlike in our own days) at the forefront of knowledge, and its claims were so many combative statements, which impacted on society at large. The broader, public significance of a discipline usually perceived today as arcane, is one of the themes I hope to show- case here. The modern study of religion developed throughout the 19th century, up to the establishment, in its last third, of the first academic chairs, journals, and conferences exclusively devoted to the new discipline. 2 On early Christian taxonomies of religions, see Francesco Massa, Nommer et classer les religions Guy G. Stroumsa The Study of Religion and the Spirit of Orientalism: Cultural Transformations and Scholarly Shifts To a great extent, it focused then on the religious systems of newly discovered peoples, throughout Asia, in the Americas, and in Africa, as well as on those from Mediterranean and Near Eastern Antiquity, which were now analyzed thanks to new philological tools and the sup- porting evidence brought by archaeology. In this context, the understanding of pol- ytheistic (or, as in the case of Buddhism, non-theistic) systems, approached for the first time in a non-polemical fashion, sine ira et studio, played a central role. In medieval Christian societies, both in Byzantium and in the Latin West, there existed a single taxonomy of the world’s religions.2 For a full millennium, roughly The Study of Religion and the Spirit of Orientalism 65 from the 8th to the 18th century, Christian thinkers had perceived the world as divided between four main religious families: Christianity, Judaism, Islam, and ‘the rest’, also known as ‘heathenism’. As a rule, this fourfold taxonomy did not entail the existence of a ‘Triple Alliance’ of sorts between the monotheistic traditions. Rather, polemics remained the usual medium of communication between them. Christianity (or, more precisely, its various orthodox versions) was of course the only true religion (vera religio in Augustine’s terms), while Judaism and Islam were considered to be erroneous ones (falsae religiones), one upstream and one down- stream of Christianity, as it were. From Epiphanius of Salamis to John of Damas- cus, Patristic heresiologists could even present them as heresies of sorts, Judaism being the first heresy and Islam the last – and worst – one.3 In different ways, Jews and Muslims remained the enemies of the true faith. But as monotheists, Jews and Muslims alike were recognized to be akin, in a deep sense, to the Christians, despite the enormous difference between them in Christian perception. While Jews were generally tolerated (and humiliated, as children of the devil [John 8:44]) in tradi- tional Christian societies, Muslims typically remained the enemy without.4 In modern times, the discovery of new societies and their traditional cultures demanded that this classification be abandoned, as the sole category of ‘heathen- ism’ was no longer adequate to interpret the great variety of religions in the world. When the old taxonomy was abandoned, the centuries-old family relationship (to use Wittgenstein’s metaphor) between the three monotheistic religions became significantly weakened. aux IIe–IVe siècles: la taxonomie ‘paganisme, judaïsme, christianisme’, in: Revue de l’histoire des religions 234 (2017), 689–715. 3 The transformation of Judaism into a Christian heresy is of course less intuitive than the Chris- tian perception of the earliest Islam as a heresy. But Epiphanius explicitly called Judaism a heresy, while Justinian’s rulings can be said to treat the Jews as heretics. See Guy G. Stroumsa, The Making of the Abrahamic Religions in Late Antiquity. Oxford: Oxford University Press 2015, 175–188. 4 Judaism and Islam did not always reciprocate the compliment paid to them by Christianity, as Jewish and Muslim medieval thinkers rarely considered Christianity to be monotheistic. 5 Throughout history, Ham, Noah’s third son, traditionally identified with blackness, has remained the incarnation of blacks and slaves. See Benjamin Braude, Cham et Noé. Race, esclavage et exégèse entre islam, judaïsme et christianisme, in: Annales. Histoire, Sciences Sociales 57 (2002), 93–125, as aux IIe–IVe siècles: la taxonomie ‘paganisme, judaïsme, christianisme’, in: Revue de l’histoire des religions 234 (2017), 689–715. Guy G. Stroumsa The Study of Religion and the Spirit of Orientalism: Cultural Transformations and Scholarly Shifts When William Jones, speaking in 1786 at the recently founded Calcutta Asiatic Society, announced that he had discovered similarities between Sanskrit, Greek, and Latin, he was launching a new classification of lan- guages and peoples, which would quickly become the main paradigm, beyond linguistics, for a number of disciplines, throughout the 19th century. Semites and Aryans now took the traditional place of the offspring of Shem and Japheth, two of Noah’s three sons.5 To a great extent, the Semites were imagined through the 5 Throughout history, Ham, Noah’s third son, traditionally identified with blackness, has remained the incarnation of blacks and slaves. See Benjamin Braude, Cham et Noé. Race, esclavage et exégèse entre islam, judaïsme et christianisme, in: Annales. Histoire, Sciences Sociales 57 (2002), 93–125, as Guy G. Stroumsa 66 model of the Hebrews (and the Jews; indeed, from the 1880s on, the newly-coined word ‘anti-Semitism’ never referred to anyone except the Jews), while the Greeks represented the focal model of the Aryans. Monotheism would now often be con- ceived as a characteristic of a postulated ancient Semitic religion, while polytheism, would usually be identified as pertaining to the nature of the Aryan religion. This new paradigm gave the old taxonomy the coup de grâce. New categories had to be forged, and this was one of the major tasks of the new ‘science of religion’. A Europe whose identity was perceived as torn between the Semitic roots of its religion and the Aryan nature of its languages and ethnicities saw the emergence of a new and deep ambivalence to monotheism. This ambivalence was echoing and amplifying those trends in the radical Enlightenment that had grown strongly critical of the established churches. The new European discovery of similarities between Sanskrit and almost all European languages led to the identification of families of languages and also of families of religions, in particular the Aryan and the Semitic religious families.6 Deducing religion (and ethnicity) from linguistics is, of course, a fallacy, a fact underlined toward the end of the 19th century by the French polymath Salomon Reinach in his long article ‘Le mirage oriental’ – a fallacy which, as we know, would have dramatic consequences.7 A number of significant European intellectuals now started to identify European languages and peoples as belonging to the Indo-Eu- ropean (or Indo-Germanic in German parlance) or Aryan family. 7 Salomon Reinach, Le mirage oriental, in: L’anthropologie 4 (1893), 539–579 and 699. See Chapter 10 below. For another use of this metaphor: see Louis Bertrand, La réalité et le Mirage oriental, in: Revue des Deux Mondes 48 [5e période] (1908), 139–172. Bertrand later published a novel entitled Le mirage oriental (Paris: Perrin, 1920). well as David M. Goldenberg, The Curse of Ham. Race and Slavery in Early Judaism, Christianity and Islam. Princeton/Oxford: Princeton University Press 2005. 6 Maurice Olender, The Languages of Paradise. Race, Religion, and Philology in the Nineteenth Cen- tury. Cambridge (MA): Harvard University Press 1992 [French original: Les langues du paradis. Aryens et Sémites: un couple providentiel. Paris: Gallimard/Le Seuil 1989]. Olender discusses some of the figures we shall encounter in the following chapters, such as Ernest Renan, Max Müller, and Ignaz Goldziher. well as David M. Goldenberg, The Curse of Ham. Race and Slavery in Early Judaism, Christianity and Islam. Princeton/Oxford: Princeton University Press 2005. 6 Maurice Olender, The Languages of Paradise. Race, Religion, and Philology in the Nineteenth Cen- tury. Cambridge (MA): Harvard University Press 1992 [French original: Les langues du paradis. Aryens et Sémites: un couple providentiel. Paris: Gallimard/Le Seuil 1989]. Olender discusses some of the figures we shall encounter in the following chapters, such as Ernest Renan, Max Müller, and Ignaz Goldziher. 7 Salomon Reinach, Le mirage oriental, in: L’anthropologie 4 (1893), 539–579 and 699. See Chapter 10 below. For another use of this metaphor: see Louis Bertrand, La réalité et le Mirage oriental, in: Revue des Deux Mondes 48 [5e période] (1908), 139–172. Bertrand later published a novel entitled Le mirage oriental (Paris: Perrin, 1920). well as David M. Goldenberg, The Curse of Ham. Race and Slavery in Early Judaism, Christianity and slam. Princeton/Oxford: Princeton University Press 2005. Guy G. Stroumsa The Study of Religion and the Spirit of Orientalism: Cultural Transformations and Scholarly Shifts They were loath to consider their own religion, Christianity, as related in any significant way to Judaism and Islam, the main extant Semitic religions, and preferred to see in it a religious expression of the Aryan race. To be sure, one should not overstate the global importance of pan-Aryanism and imagine that it represented the leading trend in 19th-century European thought. The majority among their contemporar- ies did not take the most vociferous heralds of this kind of racialism seriously. To deduct the early nature of racialist thought patterns from its radical, murderous The Study of Religion and the Spirit of Orientalism 67 consequences in the 20th century would amount to teleological reasoning. Never- theless, in the academic study of religion the Aryan-Semite taxonomy did have a major formative role.8 Paradoxically or not, Christianity itself remained relatively peripheral to the development of the new discipline. It is always easier to develop the distance needed for an objective and critical approach toward the religions of others, rather than toward one’s own. Indeed, while the traditional reflection on religion had been the object of theology and philosophy, the modern study of religion grew mainly at the interface of philology, oriental studies, and ethnology. The religion of Biblical Israel stayed within the purview of theology, while post-biblical and Rabbinic Judaism were usually considered of little interest, reflecting the religious decadence that in Christian perception had occurred among the Hebrews after the biblical prophets, and certainly since Jesus. Moreover, the historical study of Christianity and the crit- ical, philological approach to the Bible in our period remained largely in the hands of liberal Protestant theologians; Catholics were still forbidden by ecclesiastical authorities to deal with higher biblical criticism. The study of Islam, on the other hand, continued to be mainly in the hands of Arabists, who, for centuries, had typically displayed a disparaging attitude to Muhammad, dubbed a false prophet, and to his religion. As a result, Judaism, Chris- tianity, and Islam did not form part of the new discipline’s core. My primary goal here is to appreciate the consequences of this fact for the study of monotheism, in particular on the scholarly approaches to Judaism and Islam, two religions that eventually came to be perceived as quite alien to Christian Europe. 8 See Stefan Arvidsson, Aryan Idols. Indo-European Mythology as Ideology and Science. Chicago: Chicago University Press 2006. Guy G. Stroumsa The Study of Religion and the Spirit of Orientalism: Cultural Transformations and Scholarly Shifts Attitudes to Judaism and to Islam, however, stayed strikingly different from one another, as Judaism was perceived as the more intimate enemy, the enemy within, as it were, while Islam remained essentially foreign, extraneous. Moreover, in most countries of Western Europe, there existed numerous old Jewish communities, which were being emancipated in the 19th century. There were very few Muslims then, and no Muslim communities in those areas. This discrepancy also partly explains the difference in the intensity of attitudes towards Judaism and Islam. In contradistinction to the old, abstract history of ideas, intellectual history seeks to understand ideas within their full social and political context. When dealing with approaches to religion in the 19th century, accelerated secularization, growing nationalism, and imperial colonialism provide the immediate context. The analysis of scholarly discourse on religion must reflect the new status of religion in societies that were undergoing intensive processes of secularization. On the one Guy G. Stroumsa 68 hand, with the industrial revolution and the growth of cities, the working classes were learning to free themselves from ecclesiastical control, and the traditional or established churches were losing their customary grip on Western European societies. On the other hand, a number of powerful thinkers, pursuing the radical Enlightenment’s onslaught on traditional Christianity, were in search of new forms of spirituality. From Hegel’s age of the Spirit to Auguste Comte’s religion of the future, their proposals seduced many, such as Friedrich David Strauss in Germany or Edgar Quinet in France. The second half of the century saw important decline in the number of church goers, together with the growth of the historical and compar- ative study of religion.9 Expressions of religion were regressing from the public sphere to the private one. The Christian dimension of European identities, of course, was in no way dis- appearing. Rather, the semiotic range of Christianity moved, as it was increasingly transformed from the expression of Europe’s core religious identity to representing a central element of European cultural memory. This was perhaps nowhere man- ifested as clearly as in the case of the Bible in Protestant countries, where, in the 19th century, it became in its vernacular translations, a core element of education and culture, of what the Germans call Bildung.10 Even though Christianity did not always represent actual shared beliefs and practices, it certainly remained at the core of historical consciousness and national identity. 9 For an analytical description of the period, see Owen Chadwick, The Secularization of the Europe- an Mind in the Nineteenth Century. Cambridge: Cambridge University Press 1975. 10 See Jonathan Sheehan, The Enlightenment Bible. Translation, Scholarship, Culture. Princeton: Princeton University Press 2005. Cf. Chapter 3, note 8. 11 See for instance Jean-Claude Monod, La querelle de la sécularisation de Hegel à Blumenberg. Paris: Vrin 2002. On the relationship between philosophical and theological perceptions of secular- ization, see Hans Blumenberg, Säkularisierung und Selbstbehauptung. Frankfurt: Suhrkamp 1974. Guy G. Stroumsa The Study of Religion and the Spirit of Orientalism: Cultural Transformations and Scholarly Shifts This is what Hegel referred to as the transformation, or Aufhebung of Christianity in the full-fledged Geist.11 Secularization alone, however, does not explain the transformation of the status of Christianity in European consciousness from representing Europe’s reli- gious capital to its cultural capital – to use a concept crafted by the French soci- ologist Pierre Bourdieu. Both the rapid growth of nationalism and the expansion of colonial conquests in the age of imperialism must be factored in. The growing feelings of European superiority versus Asian and African peoples and cultures would, in the second half of the century, in the heyday of colonialism, be expressed in a new key, that of ‘scientific racism’. The new expression of disparaging attitudes to foreign peoples and their cultures would have an immediate, potent, and lasting impact upon scholarly conceptions. Among others, Martin Bernal, focusing on the The Study of Religion and the Spirit of Orientalism 69 study of Greek antiquity, has shown how the categories forged in 19th century scholarship reflected racist ideas.12 One paradoxical and insidious consequence of this distantiation from Judaism and Islam led to a major fracture in European identity and to a new crisis of Euro- pean consciousness, following that of the Enlightenment, so masterly analyzed by Paul Hazard.13 The depth of this fracture, as well as its effect on the status of Judaism and Islam in the European mind, has yet to be fully measured.14 Before the Enlightenment, the Near East (then usually referred to as simply ‘the East’), was considered to be the soil of all human religious origins. All religions had come from the Ancient Near East, from Egypt to Babylonia, through the lands of the Bible.15 As Christianity was perceived, essentially, as the European religion par excellence, Judaism and Islam were, for the European mind, the two surviv- ing religions from the Ancient Near East. Now, however, they became perceived only as coming from the East of the West, or as the West of the deep and true East, that of India and China. Judaism and Islam thus fell between Europe and India, between the two poles of Indo-European cultural and religious creativity. To be sure, scholars recognized that the geographical spread of Islam throughout the world was impressive, and that both Jews and Muslims believed in one God. 12 Martin Bernal, Black Athena. The Afroasiatic Roots of Classical Civilization. Volume I: The Fab- rication of Ancient Greece 1785–1985. New Brunswick (NJ): Rutgers University Press 1987; Bernal has insisted on the major role played by the intensification of racism and the central importance of ‘ethnicity’ as a principle of historical explanation. He was able to show the crucial part they played in the formation of new taxonomies opposing ‘Aryans’ to ‘Semites’. 13 Paul Hazard, The Crisis of the European Mind. 1680–1715. New York: New York Review of Books 2013 [French original: La crise de la conscience européenne. 1680–1715. Paris: Fayard 1961 [1935]]. 14 Markus Messling, Gebeugter Geist. Rassismus und Erkenntnis in der modernen europäischen Phi lologie. Göttingen: Wallerstein 2016. 15 See for instance Guy G. Stroumsa, John Selden et les origines de l’orientalisme, in: Quentin Epron (ed.), John Selden: juriste européen, Annuaire de l’Institut Michel Villey (2012), 1–11. 13 Paul Hazard, The Crisis of the European Mind. 1680–1715. New York: New York Review of Books 2013 [French original: La crise de la conscience européenne. 1680–1715. Paris: Fayard 1961 [1935]]. 14 Markus Messling, Gebeugter Geist. Rassismus und Erkenntnis in der modernen europäischen Phi- lologie. Göttingen: Wallerstein 2016. 15 See for instance Guy G. Stroumsa, John Selden et les origines de l’orientalisme, in: Quentin Epron (ed ) John Selden: juriste européen Annuaire de l’Institut Michel Villey (2012) 1 11 12 Martin Bernal, Black Athena. The Afroasiatic Roots of Classical Civilization. Volume I: The Fab- rication of Ancient Greece 1785–1985. New Brunswick (NJ): Rutgers University Press 1987; Bernal has insisted on the major role played by the intensification of racism and the central importance of ‘ethnicity’ as a principle of historical explanation. He was able to show the crucial part they played in the formation of new taxonomies opposing ‘Aryans’ to ‘Semites’. 13 Paul Hazard, The Crisis of the European Mind. 1680–1715. New York: New York Review of Books 2013 [French original: La crise de la conscience européenne. 1680–1715. Paris: Fayard 1961 [1935]]. 14 Markus Messling, Gebeugter Geist. Rassismus und Erkenntnis in der modernen europäischen Phi- lologie. Göttingen: Wallerstein 2016. 15 See for instance Guy G. Stroumsa, John Selden et les origines de l’orientalisme, in: Quentin Epron (ed.), John Selden: juriste européen, Annuaire de l’Institut Michel Villey (2012), 1–11. Guy G. Stroumsa The Study of Religion and the Spirit of Orientalism: Cultural Transformations and Scholarly Shifts Yet, all in all, racial prejudice against the Jews (distinct from traditional religious anti-Sem- itism) and condescending attitudes to Islam and Islamic societies entailed a strong devaluation of both Judaism and Islam and a clear preference for Indo-European religious systems and cultural traditions over Semitic religions and cultures. Euro- pean Christians were thus contriving to escape the Jewish, Near-Eastern origins of their religion. The close relationship of Christianity with European culture, it was argued, did not deny its universal nature, and it was therefore Europe’s duty to promote Christianity throughout Asia and Africa, along the model of its earlier conquests in the Americas. 70 Guy G. Stroumsa 70 Intellectual perceptions of Judaism and Islam, moreover, are directly related to social attitudes toward Jews and Muslims in European societies – a point which there is surely no need to belabor. In the early stages of Jewish emancipa- tion (a process that had started with the French Revolution), Jewish communities in Western European societies were being transformed very fast. Together with emancipation and less exclusion from society at large came new tensions. The tra- ditional forms of Christian anti-Judaism, which, of course, had not disappeared, were reactivated, as it were, in a new, racial key, and fresh forms of prejudice were formulated. In various ways, the Jews were now felt to be more alien than at any previous time. Their identity was perceived as an Asian one. In other words, the Jews did not really belong to Europe. Thus Herder, and so many after him. At the same time, Islam was identified as the religion of Europe’s immediate and scorned neighbor, the Ottoman Empire, and that of colonized peoples, from the Maghreb to the Indian subcontinent and beyond. As a consequence, Muslims were often despised and hated. For generations, there had existed, of course, sig- nificant Muslim communities on European soil, mainly in the Balkans. In southern European imagery, they were often pictured as peaceful traders. More often than not, however, they remained marginalized in Western European perception. Impe- rialism and colonialism, in Africa, the Near East, and South East Asia, could only strengthen negative attitudes toward Islam and Muslims and reactivate existing prejudices. Orientalist trends in art and literature, as we know, highlighted such negative attitudes towards Islam and Muslims in the 19th century. 16 Edgar Quinet, Du génie des religions. Paris: Charpentier 1842, 65–77. A century later, the literary scholar Raymond Schwab would use the expression as the title of his masterpiece, La Renaissance orientale. Paris: Payot 2014 [English translation: The Oriental Renaissance. Europe’s Rediscovery of India and the East, 1680–1880. New York: Columbia University Press 1984]. 17 Edward Said, Orientalism. New York: Random House 1978. 18 Cf. Robert Irwin, For Lust of Knowing. The Orientalists and their Enemies. London: Penguin 2006, 3–5. On the vitriolic polemics to which the book gave birth, see for instance the exchange between Said and two distinguished Arabists and Islamic scholars, Oleg Grabar and Bernard Lewis, 18 Cf. Robert Irwin, For Lust of Knowing. The Orientalists and their Enemies. London: Penguin 2006, 3–5. On the vitriolic polemics to which the book gave birth, see for instance the exchange between Said and two distinguished Arabists and Islamic scholars, Oleg Grabar and Bernard Lewis, Guy G. Stroumsa The Study of Religion and the Spirit of Orientalism: Cultural Transformations and Scholarly Shifts In the mid-19th century, the French historian and philosopher Edgar Quinet coined the phrase ’la Renaissance orientale’, by which he meant the new European scholarly interest in, and cultural sensitivity for, the civilizations of Asia.16 Since Quinet’s days, European perceptions of the Orient have remained a highly loaded topic, as demonstrated by the fate of Edward Said’s Orientalism, a book published more than a generation ago, which soon became a cult book.17 Over the years since its publication, polemics around it, at times in stringent tones, seem to remain endemic. There is no need to rehash either the important points it was making, or the detailed criticism of its various shortcomings here.18 One of the deplorable The Study of Religion and the Spirit of Orientalism 71 consequences of Said’s book, however, is of direct import to our topic, as it derided the colossal effort made by many bright, studious, and courageous scholars. They succeeded in opening new vistas to whole civilizations far beyond the borders of Europe. Intellectual curiosity, among these scholars, seems to have been boundless. A number of excellent monographs on aspects of 19th-century Orientalism have done much to highlight its rich and complex history, as well as the many links, both obvious (starting with linguistic demands) and implicit, between Orientalism and the study of religion. While the study of Islam and that of Judaism form part of the Orientalist enter- prise (and I use this word without its usual pejorative connotations), they obviously also belong to the study of religion. Like Orientalism, the latter grew throughout the 19th century, eventually becoming a full-fledged scholarly discipline. How and when was the modern, critical study of religion born? Among the branches of humanistic scholarship, the study of religion seems to have particularly suffered from a lack of reflexivity upon its own history.19 In the last generation, however, sophisticated histories of the history of religion have done much to remedy this sore state of affairs, shedding new light on the history of the modern study of religion. It is but natural that these books usually focus on the second half of the 19th century, the period when the first university chairs and scholarly journals were established in a number of European countries. In countries of Protestant culture in particu- lar, the study of religion became established in theological faculties. 20 Jonathan Z. Smith, Drudgery Divine. On the Comparison of Early Christianities and the Religions of Late Antiquity. Chicago: University of Chicago Press 1990. 21 This is to be distinguished from the point made by Philippe Borgeaud Aux origines de l’histoire in The New York Review of Books (issue of August 12, 1982). Quite oddly, the polemics continues unabated, more than forty years after the publication of Orientalism. See for instance Adam Shatz, ‘Orientalism,’ Then and Now, in: The New York Review (20.05.2019), URL: https://www.nybooks. com/daily/2019/05/20/orientalism-then-and-now/ (21.07.2022). 19 Robert Orsi, The ‘So-Called History’ of the Study of Religion, in: Method and Theory in the Study of Religion 20 (2008), 134–138: ‘The past of religious studies has been until recently largely invisi- ble…’ (134). 20 Jonathan Z. Smith, Drudgery Divine. On the Comparison of Early Christianities and the Religions of Late Antiquity. Chicago: University of Chicago Press 1990. 21 This is to be distinguished from the point made by Philippe Borgeaud, Aux origines de l’histoire des religions. Paris: Seuil 2004, 18, on religions representing the unconscious of civilizations. y 19 Robert Orsi, The ‘So-Called History’ of the Study of Religion, in: Method and Theory in the Study of Religion 20 (2008), 134–138: ‘The past of religious studies has been until recently largely invisi ble…’ (134). 21 This is to be distinguished from the point made by Philippe Borgeaud, Aux origines de l’histoire des religions. Paris: Seuil 2004, 18, on religions representing the unconscious of civilizations. 20 Jonathan Z. Smith, Drudgery Divine. On the Comparison of Early Christianities and the Religions of Late Antiquity. Chicago: University of Chicago Press 1990. 21 This is to be distinguished from the point made by Philippe Borgeaud, Aux origines de l’histoire des religions Paris: Seuil 2004 18 on religions representing the unconscious of civilizations in The New York Review of Books (issue of August 12, 1982). Quite oddly, the polemics continues unabated, more than forty years after the publication of Orientalism. See for instance Adam Shatz, ‘Orientalism,’ Then and Now, in: The New York Review (20.05.2019), URL: https://www.nybooks. com/daily/2019/05/20/orientalism-then-and-now/ (21.07.2022). 19 Robert Orsi, The ‘So-Called History’ of the Study of Religion, in: Method and Theory in the Study of Religion 20 (2008), 134–138: ‘The past of religious studies has been until recently largely invisi- ble…’ (134). in The New York Review of Books (issue of August 12, 1982). Quite oddly, the polemics continues unabated, more than forty years after the publication of Orientalism. See for instance Adam Shatz ‘Orientalism,’ Then and Now, in: The New York Review (20.05.2019), URL: https://www.nybooks com/daily/2019/05/20/orientalism-then-and-now/ (21.07.2022). Guy G. Stroumsa The Study of Religion and the Spirit of Orientalism: Cultural Transformations and Scholarly Shifts One of the major consequences of this situation is that the study of religion long remained deeply embedded in theological conceptions, as Jonathan Z. Smith was able to show regarding the religions of late antiquity and the world of Early Christianity.20 What is needed is a critical genealogy of scholarly discourse. In a way, such a genealogy would attempt to unveil the unconscious of the discipline.21 By ‘uncon- scious’, I refer to implicit, hidden principles that dictate research and the devel- opment of the field. It is no accident that such principles usually remain unex- 72 Guy G. Stroumsa 72 pressed, in the field of religion, perhaps, more than anywhere else. If Freud is right in arguing that religion is particularly fraught with repression (Verdrängung), the same may also be true of its study. Focusing on the history of scholarship, then, I seek to epitomize the study of an absence. Why is it that 19th-century religious scholarship neglected the compara- tive study of Judaism, Christianity, and Islam – a form of study that it had inherited from a long tradition? It is true that 19th-century historicism sought to see phenom- ena as rooted in their cultural and historical context and to give each nation its due, as Ranke noted. Still, comparative scholarship blossomed, in various fields, in the last decades of the century, at the acme of British imperial power. In a number of ways, the colonial enterprise fostered a comparative approach – with the avowed view of claiming the supremacy of European culture over that of other civiliza- tions. The inquiry also stands at the confluence of different disciplines blooming in our period, in particular the study of Islam on the one hand (together with Arabic, Turkic, and Iranian philology) and Jewish Studies (or rather the Wissenschaft des Judentums) on the other hand.22 Jewish scholars played a specific role in the emergence of the history of reli- gion, at the crossroads with Orientalism, in the second half of the 19th century. This role reflects their peculiar status and self-perception, as well as a unique aspect of the discipline. By and large, among European scholars of religion, only the Jews did not identify as Christians, at least culturally. Jewish scholars sought at once, in different ways, to achieve various goals, not always compatible with one another. 22 See for instance Christian Wiese, Challenging Colonial Discourse. Jewish Studies and Protestant Theology in Wilhelmine Germany. Leiden/Boston: Brill 2005. 23 A similar phenomenon can also be observed about religions and societies of black Africa, as demonstrated by David Chidester, Empire of Religion. Imperialism and Comparative Religion. Chica- go/London: Chicago University Press 2014. 24 See for instance the discussion of this theme in John Efron, German Jewry and the Allure of the Sefardic. Princeton/Oxford: Princeton University Press 2016. This embrace of the Orient would be echoed in the Zionist urge to return to the East, to Palestine. At the turn of the century, art in Jewish Palestine, too, would embrace the Orientalizing trend. 25 See Guy G. Stroumsa, The Idea of Semitic Monotheism, Chapter 9. Guy G. Stroumsa The Study of Religion and the Spirit of Orientalism: Cultural Transformations and Scholarly Shifts They sought to apply philological and historical methods to Jewish texts and docu- ments which they considered unduly ignored or misunderstood by Christian schol- ars. Moreover, moved as they were at once by a sense of belonging to their own tra- ditional Jewish culture and by their intense desire to become full-fledged members of society at large, they believed that, as Orientals living in the West, they could offer a bridge of sorts between the cultures and languages of Asia and Europe. The rise of Romanticism and of national movements contributed to the break-up of the integrative reflection on the three great monotheist traditions we now call the Abrahamic religions. This break-up was finalized by the combined impact of a traditional contempt for Islam and a patronizing (or worse) attitude to contemporary Muslim societies.23 73 The Study of Religion and the Spirit of Orientalism The Study of Religion and the Spirit of Orientalism Side by side with this aversion to Islam, the new racial anti-Semitism was growing. At the time, the Jews of Europe were starting to leave the ghettos and enter Western European societies – a dramatic change of the old patterns of relationship between Christians and Jews during many centuries. With their new economic inte- gration in society at large, however, the Jews soon discovered, painfully, that this was not enough to earn them what Heine called an ‘entrance ticket’ to European society – a ticket which only baptism could really provide. The Jews, as already mentioned, were perceived as stemming from the Orient and often considered as still belonging to it. It should be noted that Jews often embraced these oriental roots with pride. This self-identification is reflected in the Orientalizing architecture of many 19th century synagogues, a style meant to allude to the mythical symbiosis, or convivencia, between Muslims, Jews, and Christians in al-Andalus, medieval Islamic Spain.24 Side by side with this aversion to Islam, the new racial anti-Semitism was growing. At the time, the Jews of Europe were starting to leave the ghettos and enter Western European societies – a dramatic change of the old patterns of relationship between Christians and Jews during many centuries. With their new economic inte- gration in society at large, however, the Jews soon discovered, painfully, that this was not enough to earn them what Heine called an ‘entrance ticket’ to European society – a ticket which only baptism could really provide. The Jews, as already mentioned, were perceived as stemming from the Orient and often considered as still belonging – a ticket which only baptism could really provide. The Jews, as already mentioned, were perceived as stemming from the Orient and often considered as still belonging to it. It should be noted that Jews often embraced these oriental roots with pride. This self-identification is reflected in the Orientalizing architecture of many 19th century synagogues, a style meant to allude to the mythical symbiosis, or convivencia, between Muslims, Jews, and Christians in al-Andalus, medieval Islamic Spain.24 – a ticket which only baptism could really provide. The Jews, as already mentioned, were perceived as stemming from the Orient and often considered as still belonging to it. It should be noted that Jews often embraced these oriental roots with pride. 74 Guy G. Stroumsa 74 Guy G. Stroumsa 74 Guy G. Stroumsa 74 Since their birth in the 17th century, the modern humanistic disciplines, like the natural sciences, have been fascinated by comparison. The comparison between languages, cultures, legal systems, scientific traditions, mythologies, and societies, however, has always reflected an effort in detecting differences as much as in seeking similarities between them. These early modern humanistic disciplines were the offspring of the puzzlement generated by the new cultures and societies revealed by the great discoveries. The comparative element gathered momentum in the 19th century, starting with linguistics, under the impact of Franz Bopp’s seminal studies on the grammar of the Indo-European languages, and reached its zenith toward the end of that century.26 Comparison lies at the center of the non-theological study of religions since the 18th century. Ever present, and indispensable, comparison is always highly prob- lematic as a method. It is rarely innocent.27 It is also a constant exercise, one that is at the core of experimental investigation. What do we do when we compare? The answer depends, of course, upon one’s goals, viewpoint, and culture. Anthropolo- gists, in particular, have reflected much on the question of comparison between societies, both those broadly similar and those highly different from one another.28 27 Bruce Lincoln, Theses on Comparison, in: Bruce Lincoln, Apples and Oranges. Explorations In, On, and With Comparison. Chicago/London: Chicago University Press 2018, 25–33, esp. 25. See also, in the same volume, The Future of History of Religions, 14–24 (written together with Cristiano Grottanelli). David Chidester, Empire of Religion. Imperialism and Comparative Religion. Chicago/ London: Chicago University Press 2014. Chidester shows how British Imperialism in the second half of the 19th century used taxonomies of religions in order to support its colonial conquests in South Africa. See already David Chidester, Savage Systems. Colonialism and Comparative Religion in Southern Africa. Charlottesville/London: University Press of Virginia 1996. On comparison in the study of ancient religions, see Claude Calame/Bruce Lincoln (eds.), Comparer en histoire des religions antiques. Liège: Presses universitaires de Liège 2012. For the wise remarks of a Western Medievalist puzzled by what she sees in India, see Caroline Bynum, Avoiding the Tyranny of Mor- phology; Or, Why Compare?, in: History of Religions 53 (2014), 341–368. For an attempt to negotiate a path between too much and too little comparison in the study of religion, see Wendy Doniger, The Implied Spider. 26 See further, Stroumsa, Semitic Monotheism, Chapter 9. The Study of Religion and the Spirit of Orientalism This self-identification is reflected in the Orientalizing architecture of many 19th century synagogues, a style meant to allude to the mythical symbiosis, or convivencia, between Muslims, Jews, and Christians in al-Andalus, medieval Islamic Spain.24 The scholarly study of Christianity (in particular early and late antique Chris- tianity) which slowly sought to disengage itself from theology (without ever fully succeeding in achieving this goal) represents a special case in the emerging com- parative and historical study of religions. The appearance, in the last three decades of the 19th century, of the new concept of ‘world religions’ broke the traditional family relationship of Judaism, Christianity, and Islam, as it insisted in seeing Chris- tianity to be a full-fledged universalist religion, while Islam was granted this status only grudgingly, and Judaism rejected into the category of racially and ethnically determined religions.25 The history of scholarship represents more than the list of the achievements of individual scholars; it is also made of scholarly institutions, in the framework of which free research and intellectual breakthroughs can happen. Such schol- arly institutions include universities but also theological seminaries, scientific academies, scholarly journals, conferences, and publishing venues, and compa- nies. In contradistinction to early modernity, when scholarship remained essen- tially the personal adventure of highly gifted and idiosyncratic individuals, from the 19th century on, research has mainly been carried on within universities. It is, indeed, the dialectical interaction between individual thinking and institutional- ized systems of knowledge that transforms disciplines. One cannot really under- stand intellectual discourse and scholarly practices without constant reference to their cultural, religious, and ideological background. 74 Guy G. Stroumsa Politics and Theology in Myth. New York: Columbia University Press 1998, 64–71. 28 About comparison as experimental method, see Philippe Borgeaud, L’histoire des religions. Gollion: Infolio 2013, 182–185. For a rich volume of studies on comparatism by historians and an- thropologists alike, see Renaud Gagné/Simon Goldhill/Geoffrey E. R. Lloyd (eds.), Regimes of Com- paratism: Frameworks of Comparison in History, Religion and Anthropology (Jerusalem Studies in Religion and Culture 24). Leiden/Boston: Brill 2018. For an analysis of the different kinds of com- paratism in anthropology, see in particular in the volume, Philippe Descola, Anthropological Com- paratisms: Generalisation, Symmetrisation, Bifurcation, 402–417. In 2019, Descola dedicated his last year of teaching at the Collège de France to the question of comparatism in anthropology. In the footsteps of E. E. Evans-Pritchard, he insists on the fact that comparison is the very essence of an- 75 The Study of Religion and the Spirit of Orientalism Contemporary historiography of our discipline typically focuses on Protestant countries in the second half of the 19th century. Philippe Borgeaud, for instance, puts his finger on a climate of secularization in Protestant countries in the 1870s, while Hans Kippenberg follows the traditional emphasis on Protestant scholar- ship.29 Until the early 20th century, the Catholic hierarchy was still fiercely fighting the critical methods in the study of the Scriptures, and Catholic theological facul- ties remained, by and large, unwilling to study religious phenomena and history in a modern, non-traditional way. Despite various daring attempts, such as those of the Dominicans of the École Biblique in Jerusalem since the days of Marie-Joseph Lagrange, O.P., Catholic scholars were prohibited from practicing higher criticism until Vatican II. More precisely, it was within Protestant theological faculties in Germany that the new critical approach to the Scriptures was born, and that the progressive the- ological liberalization was most conspicuous, permitting the study of non-Chris- tian religions, past and present. Fresh scholarly biblical hermeneutics eventually opened the way to the comparative study of religious texts from both the Ancient Near East and the Greco-Roman Mediterranean – a trend famously illustrated by the Göttingen Religionsgeschichtliche Schule in the last decade of the 19th century.30 These ancient texts were now understood as reflecting the background, or Sitz im Leben, of the religious ideas expressed in the books of the Old and New Testament. thropology and seeks to distinguish between different kinds of comparatism. In this context, it is significant that, like the study of religion, modern anthropology dates from the last decades of the 19th century. In his ‘Marett Lecture’ (1950), Evans-Prichard had already argued that anthropology should be perceived as a kind of history, and that it belonged to the Humanities. See Edward Evan Evans-Pritchard, Social Anthropology. Past and Present, in: Edward Evan Evans-Pritchard, Social Anthropology and Other Essays. New York: The Free Press 1962, 139–154, esp. 152–154. thropology and seeks to distinguish between different kinds of comparatism. In this context, it is significant that, like the study of religion, modern anthropology dates from the last decades of the 19th century. In his ‘Marett Lecture’ (1950), Evans-Prichard had already argued that anthropology should be perceived as a kind of history, and that it belonged to the Humanities. See Edward Evan Evans-Pritchard, Social Anthropology. Past and Present, in: Edward Evan Evans-Pritchard, Social Anthropology and Other Essays. New York: The Free Press 1962, 139–154, esp. 152–154. 29 Philippe Borgeaud, L’histoire des religions, 134. Kippenberg’s Discovering Religious History in the Modern Age, and is also true for those works calling attention to the high price paid by those reading religious history through confessional glasses. On this issue, Jonathan Z. Smith has shown in his seminal Drudgery Divine how much Protestant beliefs had impacted the study of early Chris- tianity in its Hellenistic background. Mutatis mutandis, a similar argument could be made about ancient Israel and Near Eastern religions. 30 On the Religionsgeschichtliche Schule, see Gerd Lüdemann (ed.), Die ‚Religionsgeschichtliche Schule‘. Facetten eines theologischen Umbruchs. Frankfurt/New York: Peter Lang 1996. See Chapter Eight below. 30 On the Religionsgeschichtliche Schule, see Gerd Lüdemann (ed.), Die ‚Religionsgeschichtliche Schule‘. Facetten eines theologischen Umbruchs. Frankfurt/New York: Peter Lang 1996. See Chapter Eight below. 29 Philippe Borgeaud, Lhistoire des religions, 134. Kippenberg s Discovering Religious History in the Modern Age, and is also true for those works calling attention to the high price paid by those reading religious history through confessional glasses. On this issue, Jonathan Z. Smith has shown in his seminal Drudgery Divine how much Protestant beliefs had impacted the study of early Chris- tianity in its Hellenistic background. Mutatis mutandis, a similar argument could be made about ancient Israel and Near Eastern religions. 74 Guy G. Stroumsa The obvious and massive cleft between Protestant and Catholic biblical schol- arship, however, is also responsible for a common error of perception, when it is too often assumed that the 19th-century study of religion is essentially a Protestant affair. The combined evidence, showing the major importance of the transmission of knowledge between different European countries, and the crucial significance 30 On the Religionsgeschichtliche Schule, see Gerd Lüdemann (ed.), Die ‚Religionsgeschichtliche Schule‘. Facetten eines theologischen Umbruchs. Frankfurt/New York: Peter Lang 1996. See Chapter Eight below 30 On the Religionsgeschichtliche Schule, see Gerd Lüdemann (ed.), Die ‚Religionsgeschichtliche Schule‘. Facetten eines theologischen Umbruchs. Frankfurt/New York: Peter Lang 1996. See Chapter Eight below. 76 Guy G. Stroumsa 76 for the science of religion of ethnological studies, coming at the time mainly from Catholic missionaries, clearly discards such a misperception. It is as a complex com- bination of elements coming from different cultural and intellectual traditions that the modern study of religion emerged.31 In the trajectory of modern scholarship on religion, a number of significant shifts in systems of knowledge brought to the reconstruction of central cognitive structures. The formation and re-structuring of concepts and methods modifies fields of study, sometimes profoundly transforming them. Such fields are ultimately related to the construction of the self, in particular when they deal directly with religious identities. In a time of profound and extensive secularization of European societies, the historical and comparative study of religions, which is a particularly delicate and interdisciplinary field of scholarship, has had a significant impact on both the transmission of knowledge and the transformation of European identities. What was true in the 19th century remains true today. We witness, through- out Europe, bursts of Islamophobia and an animated, enduring, often strident, and sometimes violent public discussion about the ethnic, cultural, and religious impli- cations of the massive immigration from Muslim countries, coming together with a worrisome renewal, under various garbs, of a Judaeophobia we naively thought was on the waning.32 Even the largely post-Christian Europe of the 21st century, indeed, does not seem to be quite done with its old obsessions about Muslims and Jews. 31 I reached a similar conclusion about the early modern study of religion, in the 17th century, in A New Science. ew Science. 31 I reached a similar conclusion about the early modern study of religion, in the 17th century, in A New Science. 32 For a number of reasons, the use of ‘Semite’ and ‘Semitic’ has continued to be deeply ambiva- lent, and to this day, is often problematic. ‘Anti-Semitism’, as we know, is a term coined in the early 1880s, by the German journalist Wilhelm Marr, naming the political movement opposing the inte- gration of Jews into society. Although this misnomer has retained its original connotations to this day, it is often argued that Arabs cannot be accused of Antisemitism in the sense of Judeophobia, since they too are Semites. This is of course a very weak syllogism, as has been demonstrated many times. Another claim, more relevant to our present inquiry, was levelled by Edward Said, when he argued that the popular anti-Semitic animus was transferred from a Jewish to an Arab target, and that this transference was made smoothly since the figure was essentially the same (i.e., the Arabs too are Semites, and therefore the same word can be used against them too). This is a specious argument, as cogently argued by Bernard Lewis in his Semites and Anti-Semites. New York/London: Norton 1986. The word itself, anti-Semitism, dates from the late 19th century, while according to Said, Orientalist anti-Arab animus is a much earlier phenomenon. Moreover, anti-Jewish animus never weakened, let alone disappeared, even with the growth of what is now called Islamophobia. Neither the word ‘anti-Semitism’ nor the hatred it refers to can be said to have been transferred from Jew to Arab. On this, see Gil Anidjar, Semites. Race, Religion, Literature. Stanford: Stanford University Press 2008, 13–38. 31 I reached a similar conclusion about the early modern study of religion, in the 17th century, in A New Science. 74 Guy G. Stroumsa 32 For a number of reasons, the use of ‘Semite’ and ‘Semitic’ has continued to be deeply ambiva- lent, and to this day, is often problematic. ‘Anti-Semitism’, as we know, is a term coined in the early 1880s, by the German journalist Wilhelm Marr, naming the political movement opposing the inte- gration of Jews into society. Although this misnomer has retained its original connotations to this day, it is often argued that Arabs cannot be accused of Antisemitism in the sense of Judeophobia, since they too are Semites. This is of course a very weak syllogism, as has been demonstrated many times. Another claim, more relevant to our present inquiry, was levelled by Edward Said, when he argued that the popular anti-Semitic animus was transferred from a Jewish to an Arab target, and that this transference was made smoothly since the figure was essentially the same (i.e., the Arabs too are Semites, and therefore the same word can be used against them too). This is a specious argument, as cogently argued by Bernard Lewis in his Semites and Anti-Semites. New York/London: Norton 1986. The word itself, anti-Semitism, dates from the late 19th century, while according to Said, Orientalist anti-Arab animus is a much earlier phenomenon. Moreover, anti-Jewish animus never weakened, let alone disappeared, even with the growth of what is now called Islamophobia. Neither the word ‘anti-Semitism’ nor the hatred it refers to can be said to have been transferred from Jew to Arab. On this, see Gil Anidjar, Semites. Race, Religion, Literature. Stanford: Stanford University Press 2008, 13–38.
https://openalex.org/W4384521808	https://hal.science/hal-04462059/document	English	null	A multilayered approach to the analysis of genetic data from individuals with suspected albinism	Journal of medical genetics	2,023	cc-by	4,989	Panagiotis I Sergouniotis, Vincent Michaud, Eulalie Lasseaux, Christopher Campbell, Claudio Plaisant, Sophie Javerzat, Ewan Birney, Simon C Ramsden, Graeme C Black, Benoit Arveiler To cite this version: Panagiotis I Sergouniotis, Vincent Michaud, Eulalie Lasseaux, Christopher Campbell, Claudio Plaisant, et al.. A multilayered approach to the analysis of genetic data from individuals with suspected albinism.. Journal of Medical Genetics, 2023, 60 (12), pp.1245-1249. 10.1136/jmg-2022-109088. hal- 04462059 Distributed under a Creative Commons Attribution 4.0 International License ABSTRACT genes remain to be discovered, alterations in such genes are expected to explain only a very small frac- tion of cases. Indeed, pathogenic variants in two genes that have been recently implicated in albi- nism, DCT and BLOC1S5, were detected in only 2 of 1500 (0.13%) albinism cases each.3 4 Albinism is a clinically and genetically heterogeneous group of conditions characterised by visual abnormalitie and variable degrees of hypopigmentation. Multiple studies have demonstrated the clinical utility of genetic investigations in individuals with suspected albinism. Despite this, the variation in the provision of genetic testing for albinism remains significant. One key issue is the lack of a standardised approach to the analysis of genomic data from affected individuals. For example, there is variation in how different clinical genetic laboratories approach genotypes that involve incompletely penetrant alleles, including the common, ’hypomorphic’ TYR c.1205G>A (p.Arg402Gln) [rs1126809] variant. Here, we discuss the value of genetic testing as a frontline diagnostic tool in individuals with features of albinism and propose a practice pattern for the analysis of genomic data from affected families. 2European Molecular Biology Laboratory, European Bioinformatics Institute (EMBL- EBI), Wellcome Genome Campus, Cambridge, UK 3Manchester Centre for Genomic Medicine, Saint Mary’s Hospital, Manchester University NHS Foundation Trust, Manchester, UK Obtaining a precise genetic diagnosis in people with albinism: ► Allows timely identification of the subset of affected individuals who are at risk of devel- oping major complications (lung fibrosis, prolonged bleeding and/or granulomatous colitis in individuals with certain subtypes of HPS; immunodeficiency and neurodegen- eration in individuals with Chediak-Higashi syndrome [CHS]). In these cases, early diag- nosis can drive evidence-based changes in care management and result in improved outcomes (a relevant example can be found in the Cases section: proband 1). 5Department of Medical Genetics, University Hospital of Bordeaux, Bordeaux, France 6INSERM U1211, Rare Diseases, Genetics and Metabolism, University of Bordeaux, Bordeaux, France ► Helps rule out differential diagnoses in individ- uals presenting with non-specific features such as nystagmus and/or foveal hypoplasia. A multilayered approach to the analysis of genetic data from individuals with suspected albinism Panagiotis I. Sergouniotis ‍ ‍ ,1,2,3,4 Vincent Michaud ‍ ‍ ,5,6 Eulalie Lasseaux,5 Christopher Campbell,3 Claudio Plaisant,5 Sophie Javerzat,6 Ewan Birney,2 Simon C. Ramsden,3 Graeme C. Black,1,3 Benoit Arveiler5,6 on February 15, 2024 at GH Pellegrin. Protected by copyright. http://jmg.bmj.com/ blished as 10.1136/jmg-2022-109088 on 17 July 2023. Downloaded from 1Division of Evolution, Infection and Genomics, School of Biological Sciences, Faculty of Biology, Medicine and Health, University of Manchester, Manchester, UK 2European Molecular Biology Laboratory, European Bioinformatics Institute (EMBL- EBI), Wellcome Genome Campus, Cambridge, UK 3Manchester Centre for Genomic Medicine, Saint Mary’s Hospital, Manchester University NHS Foundation Trust, Manchester, UK 4Manchester Royal Eye Hospital, Manchester University NHS Foundation Trust, Manchester, UK 5D t t f M di l Short report Received 1 December 2022 Accepted 12 May 2023 Published Online First 17 July 2023 HAL Id: hal-04462059 https://hal.science/hal-04462059v1 Submitted on 16 Feb 2024 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License Vision science on February 15, 2024 at GH http://jmg.bmj.com/ J Med Genet: first published as 10.1136/jmg-2022-109088 on 17 July 2023. Downloaded from 1Division of Evolution, Infection and Genomics, School of Biological Sciences, Faculty of Biology, Medicine and Health, University of Manchester, Manchester, UK 2European Molecular Biology Laboratory, European Bioinformatics Institute (EMBL- EBI), Wellcome Genome Campus, Cambridge, UK 3Manchester Centre for Genomic Medicine, Saint Mary’s Hospital, Manchester University NHS Foundation Trust, Manchester, UK 4Manchester Royal Eye Hospital, Manchester University NHS Foundation Trust, Manchester, UK BACKGROUND ► Facilitates accurate genetic counselling and can inform reproductive planning in affected indi- viduals and families. Correspondence to Panagiotis I. Sergouniotis; panagiotis.sergouniotis@ manchester.ac.uk Benoit Arveiler; benoit.arveiler@chu-bordeaux.fr Graeme C. Black; graeme.black@manchester. ac.uk Albinism is a group of conditions associated with reduced levels of melanin pigment that result in developmental visual system anomalies; skin and hair manifestations are also present in the majority of cases. Significant phenotypic variability has been described among affected individuals. The pigmen- tation of the skin, the hair and the uveal layer of the eye, for example, can range from very reduced to (almost) normal. The ophthalmic features, which are the hallmark of the condition, are also variable and can include nystagmus, reduced best-corrected visual acuity, photoaversion, iris transillumination, foveal hypoplasia and chiasmal misrouting of the optic nerves.1 To reach a genetic diagnosis, integration of phenotypic and genomic information is required; family context and dynamic changes in pigmen- tation should also be taken into account. In this article, we discuss clinical genetic testing in individ- uals who have features of albinism and propose a molecular diagnostic decision tree. GCB and BA contributed equally. For brevity, this graph focuses on autosomal recessive forms of albinism and the X-linked, GPR143-related subtype is omitted. ACMG, American College of Medical Genetics and Genomics; heteroz, heterozygous; homoz, homozygous. High-throughput DNA sequencing approaches are typically used for genetic variant detection. These approaches can be complemented by high-resolution array CGH (comparative genomic hybridization) analyses that focus on CNVs, which are thought to represent >10% of pathogenic alleles.5 It is noted that bioinformatic tools that mine sequencing data are increas- ingly replacing array CGH-based workflows. Established criteria should be used to analyse these changes. Presently, most genetic laboratories use the variant classification system developed by the ACMG-AMP (American College of Medical Genetics and Genomics and the Association for Molec- ular Pathology).9 10 This involves scoring genetic alterations and assigning them into one of the following five classes: benign (class 1), likely benign (class 2), variant of uncertain significance (VUS) (class 3), likely pathogenic (class 4) and pathogenic (class 5). Established criteria should be used to analyse these changes. Presently, most genetic laboratories use the variant classification system developed by the ACMG-AMP (American College of Medical Genetics and Genomics and the Association for Molec- ular Pathology).9 10 This involves scoring genetic alterations and assigning them into one of the following five classes: benign (class 1), likely benign (class 2), variant of uncertain significance (VUS) (class 3), likely pathogenic (class 4) and pathogenic (class 5). g y p g y Like other rare genetic disorders, molecular diagnostic prac- tice patterns for albinism rely heavily on a search for low- frequency (rare), highly penetrant genetic variants. This reflects the wide acceptance of the rare disease – rare variant hypothesis which states that, if a disorder with a significant genetic compo- nent is rare, then the underlying genetic defect(s) will also be rare. However, in the past decade, a number of studies have chal- lenged the universal nature of this paradigm and have highlighted the role of common genetic variation in rare phenotypes.6 7 In light of this and the findings of our recent work on albinism,8 we propose a three-step approach to the analysis of genetic data from patients with suspected albinism, i.e. individuals with at least one of the key ophthalmic features of the condition (for example prominent foveal hypoplasia) (figure 1). The presence of two class 4 or 5 variants in a gene of interest confirms a genetic diagnosis after phase has been established (unless the relevant gene is on the X-chromosome or it is associ- ated with an autosomal dominant inheritance pattern, in which case, one such change can be sufficient). APPROACH TO GENETIC TESTING IN ALBINISM Predictive testing, either in the context of a specific family or in broader settings requires a customised approach. Notably, as genotypes that include albinism-associated TYR haplotypes are unlikely to be fully penetrant, accurate risk prediction remains challenging. The focus of this decision tree is diagnostic genetic testing. Predictive testing, either in the context of a specific family or in broader settings requires a customised approach. Notably, as genotypes that include albinism-associated TYR haplotypes are unlikely to be fully penetrant, accurate risk prediction remains challenging. The focus of this decision tree is diagnostic genetic testing. Predictive testing, either in the context of a specific family or in broader settings requires a customised approach. Notably, as genotypes that include albinism-associated TYR haplotypes are unlikely to be fully penetrant, accurate risk prediction remains challenging. For brevity, this graph focuses on autosomal recessive forms of albinism and the X-linked, GPR143-related subtype is omitted. ACMG, American College of Medical Genetics and Genomics; heteroz, heterozygous; homoz, homozygous. Common variant analysis ergouniotis PI, et al. J Med Genet 2023;60:1245–1249. doi:10.1136/jmg-2022-109088 y Common variation in the TYR gene (encoding tyrosinase) has been implicated in relatively mild forms of albinism since the early 1990s.11–13 We have recently shown that the presence of haplotypes that include specific alleles of three common TYR Common variation in the TYR gene (encoding tyrosinase) has been implicated in relatively mild forms of albinism since the early 1990s.11–13 We have recently shown that the presence of haplotypes that include specific alleles of three common TYR For brevity, this graph focuses on autosomal recessive forms of albinism and the X-linked, GPR143-related subtype is omitted. ACMG, American College of Medical Genetics and Genomics; heteroz, heterozygous; homoz, homozygous. Variants of uncertain significance cannot contribute to a high-confidence genetic diagnosis that guides clinical decision making but are worth reporting, sharing in open-access databases (such as ClinVar) and being considered for reclassification at a later stage. at GH Pellegrin. Protected by copyright. APPROACH TO GENETIC TESTING IN ALBINISM APPROACH TO GENETIC TESTING IN ALBINISM An initial aim of genetic testing in individuals suspected of having albinism is the identification of single-nucleotide variants (SNVs) and copy number variants (CNVs) impacting albinism-related genes. Additional analysis of other genes — such as FRDM7, SLC38A8 and PAX6 — is recom- mended; although these genes have been impli- cated in distinct conditions that do not generally feature hypopigmentation, the associated pheno- typic spectra include characteristic ophthalmolog- ical manifestations of albinism. Notably, infants whose presentation is linked to changes in these three genes may have lightly pigmented skin/hair due to their familial/genomic background; this can increase the apparent overlap between these condi- tions and albinism.5 at GH Pellegrin. Protected by copyright. At least 20 genetically distinct forms of albi- nism have been described. These can be caused by variants in genes encoding: melanogenic enzymes (TYR, TYRP1 and DCT); channels regulating mela- nosomal pH (OCA2 and SLC45A2) or calcium homeostasis within melanin-containing cells (SLC24A5); subunits of multiprotein complexes that are involved in melanosome maturation and/or trafficking of melanogenic enzymes (Hermansky- Pudlak syndrome [HPS] group of genes).2 Most forms of albinism are inherited as autosomal reces- sive traits apart from GPR143-related albinism, which is an X-linked recessive condition. Although it is possible that additional albinism-associated 1245 Sergouniotis PI, et al. J Med Genet 2023;60:1245–1249. doi:10.1136/jmg-2022-109088 Vision science on February 15, 2024 at GH Pellegrin. Protected by copyright. http://jmg.bmj.com/ J Med Genet: first published as 10.1136/jmg-2022-109088 on 17 July 2023. Downloaded from Vision science Figure 1 Schematic showing the proposed workflow for downstream analysis of genetic variants detected in individuals who have features of albinism. p j g y on February 15, 2024 http://jmg.bmj.com/ J Med Genet: first published as 10.1136/jmg-2022-109088 on 17 July 2023. Downloaded from Figure 1 Schematic showing the proposed workflow for downstream analysis of genetic variants detected in indivi Figure 1 Schematic showing the proposed workflow for downstream analysis of genetic variants detected in individuals who have features of albinism. The focus of this decision tree is diagnostic genetic testing. Predictive testing, either in the context of a specific family or in broader settings requires a customised approach. Notably, as genotypes that include albinism-associated TYR haplotypes are unlikely to be ful penetrant, accurate risk prediction remains challenging. The focus of this decision tree is diagnostic genetic testing. Obtaining phase information This approach is imperfect as obtaining appropriate samples from relatives can be challenging; the analysis may uncover misattributed genetic relationships; testing of multiple individuals over more than one generation may be required to obtain fully informative genotypes. ► TYR c.[−301C;575C>A;1205G>A]; this can also be described as TYR c.[−301=;575C>A;1205G>A] or [C;A;A]; the frequency of this haplotype in people with European-like ancestries is ~1%. ► TYR c.[−301C;575C;1205G>A]; this can also be described as TYR c.[−301=;575=;1205G>A] or [C;C;A]; the frequency of this haplotype in people with European-like ancestries is ~0.3%. on February 15, 2024 at GH Pellegr http://jmg.bmj.com/ 88 on 17 July 2023. Downloaded from It is recommended to seek these two rare haplotypes in the genetic data of individuals with suspected albinism who carry the TYR c.1205G>A change. Current evidence suggests that the presence of these haplotypes, either in the homozygous state or in trans to a TYR class 4 or 5 variant, would be sufficient to establish the genetic diagnosis, especially in people who have relatively mild forms of albinism (a relevant example can be found in the Cases section: proband 2). It is highlighted that the TYR c.-301C and the TYR c.575A (p.Ser192Tyr) alleles are frequently found in linkage disequilibrium so that the presence In the context of albinism, phase information can help to (i) identify compound heterozygosity (for example, when two rare variants are detected) or (ii) to clarify the haplotypic struc- ture around the TYR locus when common albinism-associated changes are present (a relevant example can be found in the Cases section: proband 3). In these scenaria, phasing can be key to obtaining a high-confidence genetic diagnosis. Figure 2 Example of how the analysis of a common TYR promoter variant can help obtain a genetic diagnosis in people with albinism (see also Cases section: proband 3). It is highlighted that the TYR c.-301C allele is predicted to reduce TYR gene expression in fetal retinal pigment epithelia by altering an OTX2 binding site in the TYR promoter.16 17 In contrast, the TYR c.1205G>A missense change produces a temperature-sensitive enzyme that has reduced catalytic activity and a tendency to be retained in the endoplasmic reticulum.11 17–20 Notably, the TYR c.-301 position is unlikely to be targeted by many currently used genomic assays (including certain panel-based and exome sequencing tests), and expansion of the target enrichment design to include this region may be required. on February 15, 2024 at GH Pellegrin. Rare variant analysis d d h It is recommended that the initial focus remains on the detec- tion and classification of rare SNVs and CNVs in relevant genes. Sergouniotis PI, et al. J Med Genet 2023;60:1245–1249. doi:10.1136/jmg-2022-109088 1246 Vision science on February 15, 2024 at GH Pellegrin. Protected http://jmg.bmj.com/ J Med Genet: first published as 10.1136/jmg-2022-109088 on 17 July 2023. Downloaded from of the latter would, in most cases, suggest the presence of the former on the same haplotype. of the latter would, in most cases, suggest the presence of the former on the same haplotype. variants has similar effects to rare sequence alterations that have high penetrance.8 These three common variants are variants has similar effects to rare sequence alterations that have high penetrance.8 These three common variants are variants has similar effects to rare sequence alterations that have high penetrance.8 These three common variants are ► TYR c.-301C>T [rs4547091], located in the promoter, with an allele frequency of ~60% (for the effect allele which can be described as c.-301C or c.-301) in non-Finnish European (NFE) populations in the Genome Aggregation Database (gnomAD v2.1.1). Obtaining phase information At present, the data generated by most DNA sequencing work- flows take the form of unphased genotypes. As a result, it is generally not possible to directly observe on which of the two parental chromosomes (or haplotypes) a particular allele falls on. To address this, haplotype phasing must be performed in order to assign genetic variants to the corresponding paternal or maternal chromosomes. on February 15, 2024 at GH Pellegrin. Protected by copyright. http://jmg.bmj.com/ first published as 10.1136/jmg-2022-109088 on 17 July 2023. Downloaded from http://jmg.bmj.com/ published as 10.1136/jmg-2022-109088 on 17 July 2023. Downloaded from ► TYR c.575C>A (p.Ser192Tyr) [rs1042602], with a minor allele frequency of ~36% in gnomAD NFE populations. ► TYR c.575C>A (p.Ser192Tyr) [rs1042602], with a minor allele frequency of ~36% in gnomAD NFE populations. ► TYR c.1205G>A (p.Arg402Gln) [rs1126809], with a minor allele frequency of ~27% in gnomAD NFE populations. The relevant albinism-associated haplotypes are Typically, the focus of haplotype phasing is heterozygous geno- types. Two main phasing methods have been described: mendelian phasing (which uses parental genotypes) and statistical phasing (which uses intermarker correlation). More recently, advances in long-read sequencing technologies have enabled phasing without relying on statistical inference or testing relatives.14 Nonetheless, the most commonly used phasing method in clinical practice at present involves analysing parental samples. This approach is imperfect as obtaining appropriate samples from relatives can be challenging; the analysis may uncover misattributed genetic relationships; testing of multiple individuals over more than one generation may be required to obtain fully informative genotypes. In the context of albinism, phase information can help to (i) identify compound heterozygosity (for example, when two rare variants are detected) or (ii) to clarify the haplotypic struc- ture around the TYR locus when common albinism-associated changes are present (a relevant example can be found in the Cases section: proband 3). In these scenaria, phasing can be key to obtaining a high-confidence genetic diagnosis. Typically, the focus of haplotype phasing is heterozygous geno- types. Two main phasing methods have been described: mendelian phasing (which uses parental genotypes) and statistical phasing (which uses intermarker correlation). More recently, advances in long-read sequencing technologies have enabled phasing without relying on statistical inference or testing relatives.14 Nonetheless, the most commonly used phasing method in clinical practice at present involves analysing parental samples. CASES in the analysis and the key role of haplotype phasing are highlighted. Three cases are discussed further to illustrate the utility of genetic testing in albinism (proband 1) and to highlight the value of the proposed molecular diagnostic approach (probands 2 and 3). Our work in particular clarifies the long-lasting debate about the impact of the TYR c.1205G>A (p.Arg402Gln) variant and its definition as a benign or pathogenic change.11–13 We argue that this variant can be considered to be pathogenic/predisposing in the context of specific haplo- types/genotypes, but not on its own. p p g pp (p ) Proband 1 was diagnosed with oculocutaneous albinism in the first few months of life. He presented with nystagmus and was found to have iris transillumination, fundal hypopigmenta- tion, and a featureless fovea. His skin and hair were moderately pigmented and there were no other concerns about his health/ development. There was no family history of albinism or visual problems. Genetic testing was requested and revealed a homo- zygous HPS5 c.1507G>T (p.Glu503Ter) variant. Pathogenic variants in HPS5 are known to cause a form of HPS that is characterised by albinism and dysfunction of blood platelets (leading to prolonged bleeding). The infant was referred to the paediatric haematology department where he had platelet function analysis. This revealed prolonged PFA-100 closure time following stimulation with collagen and epinephrine (>300 s, reference range 79–205 s). This finding had signifi- cant implications as the child was due to have a routine urolog- ical procedure, which was safely performed with appropriate precautions. Notably, in our cohorts, approximately 1 in 20 individuals with suspected albinism is found to have biallelic pathogenic alterations in HPS-related or CHS-related genes.5 This case is also discussed in a previous report by our group where the benefits of genetic testing over more invasive and labour-intensive haematological approaches for the diagnosis of HPS are highlighted.15 http://jmg.bmj.com/ ublished as 10.1136/jmg-2022-109088 on 17 July 2023. Downloaded from In our setting, incorporating the two complex albinism- associated TYR haplotypes ([C;A;A] and [C;C;A]) in the analysis increased the diagnostic yield of genetic testing by 19%. This highlights the significant contribution of these changes to the genetic architecture of albinism. It can be speculated that TYR c.1205G>A (p.Arg402Gln) is not the only missense TYR change whose effect can be modi- fied by other variants. REFERENCES 1 Kruijt CC, de Wit GC, Bergen AA, et al. The phenotypic spectrum of albinism. Ophthalmology 2018;125:1953–60. 2 Bakker R, Wagstaff EL, Kruijt CC, et al. The retinal pigmentation pathway in human albinism: Not so black and white. Prog Retin Eye Res 2022;91:101091. 3 Pennamen P, Le L, Tingaud-Sequeira A, et al. BLOC1S5 pathogenic variants cause a new type of Hermansky-Pudlak syndrome. Genet Med 2020;22:1613–22. Sergouniotis PI, et al. J Med Genet 2023;60:1245–1249. doi:10.1136/jmg-2022-109088 Obtaining phase information Protected by copyrig http://jmg.bmj.com/ d from on February 15, 2024 at GH Pellegrin. Protected by copyright. tp://jmg.bmj.com/ Figure 2 Example of how the analysis of a common TYR promoter variant can help obtain a genetic diagnosis in people with albinism (see also Cases section: proband 3). It is highlighted that the TYR c.-301C allele is predicted to reduce TYR gene expression in fetal retinal pigment epithelia by altering an OTX2 binding site in the TYR promoter.16 17 In contrast, the TYR c.1205G>A missense change produces a temperature-sensitive enzyme that has reduced catalytic activity and a tendency to be retained in the endoplasmic reticulum.11 17–20 Notably, the TYR c.-301 position is unlikely to be targeted by many currently used genomic assays (including certain panel-based and exome sequencing tests), and expansion of the target enrichment design to include this region may be required. heteroz, heterozygous; homoz, homozygous. 1247 Sergouniotis PI, et al. J Med Genet 2023;60:1245–1249. doi:10.1136/jmg-2022-109088 Sergouniotis PI, et al. J Med Genet 2023;60:1245–1249. doi:10.1136/jmg-2022-109088 Vision science ORCID iDs Panagiotis I. Sergouniotis http://orcid.org/0000-0003-0986-4123 Vincent Michaud http://orcid.org/0000-0002-5788-392X CASES We expect that, in the future, genetic investigations for albinism will analyse key non-coding regions (including promoters and cis-regulatory elements) for all relevant genes. Notably, the concepts discussed here are likely to be relevant to the study of other rare disorders. on February 15, 2024 at GH Pellegr http://jmg.bmj.com/ 088 on 17 July 2023. Downloaded from Acknowledgements We thank Stephanie Barton (Manchester Centre for Genomic Medicine), David Green (University of Manchester), Claire Hardcastle (Manchester Centre for Genomic Medicine) and Tom Fitzgerald (EMBL-EBI) for the helpful discussions and insights. Provenance and peer review Not commissioned; externally peer reviewed. Provenance and peer review Not commissioned; externally peer reviewed. Open access This is an open access article distributed in accordance with the Creative Commons Attribution 4.0 Unported (CC BY 4.0) license, which permits others to copy, redistribute, remix, transform and build upon this work for any purpose, provided the original work is properly cited, a link to the licence is given, and indication of whether changes were made. See: https://creativecommons.org/ licenses/by/4.0/. Patient consent for publication Not applicable Ethics approval Ethical approval was obtained from the Yorkshire and The Humber Bradford Leeds Research Ethics Committee (REC reference 15/YH/0365). Ethics approval Ethical approval was obtained from the Yorkshire and The Humber Bradford Leeds Research Ethics Committee (REC reference 15/YH/0365). Ethics approval Ethical approval was obtained from the Yorkshire and The Humber Bradford Leeds Research Ethics Committee (REC reference 15/YH/0365). p g p p g Proband 3 has a diagnosis of oculocutaneous albinism. Conven- tional genetic analysis focusing on rare variants revealed a hetero- zygous likely pathogenic variant in TYR c.823G>T (p.Val275Phe) [rs104894314]; this was found to be inherited from the proband’s mother (figure 2). When the common, functionally-relevant TYR c.1205G>A (p.Arg402Gln) variant was included in the analysis, the proband appeared to have a genotype similar to that of his unaffected mother. Analysis of the common promoter variant TYR c.-301C>T, as well as of TYR c.575C>A (p.Ser192Tyr), and testing of samples from other family members highlighted that the proband carried the likely pathogenic TYR c.823G>T change in trans to the c.[−301C;575C;1205G>A] haplotype (figure 2). This rare haplo- type is likely to be pathogenic/predisposing,8 and the fact that it is present in trans to a likely pathogenic change confirms the genetic diagnosis of albinism. Collaborators N/A. Contributors PIS, EB, GCB and BA conceived and designed the project; all other others contributed to the analysis and interpretation of data for the work. PIS drafted the manuscript; all other authors critically revised it. All authors provided final approval of the version to be published, and agreement to be accountable for all aspects of the work. Proband 2 was noted to have nystagmus soon after birth. There were no other medical concerns and there was no family history of relevance. The diagnosis of infantile nystagmus was made and the proband was followed up in his local ophthalmology unit for a number of years. An assessment in a tertiary ophthalmic genetic clinic was initiated in middle childhood, and the vision at that point was 0.3 LogMAR (Logarithm of the Minimum Angle of Resolu- tion) in each eye. His irides were blue and there were no transillu- mination defects. Optical Coherence Tomography (OCT) imaging revealed foveal hypoplasia. It is noted that the broband’s hair were light brown and his skin colour was similar to that of close relatives. Genetic testing was initiated, revealing homozygosity for the TYR c.[−301C;575C>A;1205G>A] or (C;A;A) haplotype described previously. This genotype has been shown to confer a high ‘risk of albinism’ and should be considered pathogenic/predisposing.8 Funding This study was supported by: the Wellcome Trust (224643/Z/21/Z and 200990/Z/16/Z), Retina UK and Fight for Sight (GR586 (RP Genome Project — UK IRDC), the French Albinism Association (Genespoir), the French National Research Agency (ANR-21-CE17-0041-01), and the UK National Institute for Health Research (NIHR) (CL-2017-06-001 and NIHR203308). Competing interests EB is a paid consultant and equity holder of Oxford Nanopore, a paid consultant to Dovetail, and a non-executive director of Genomics England, a limited company wholly owned by the UK Department of Health and Social Care. All other authors declare no competing interests. The views expressed are those of the authors and not necessarily those of the NIHR or the Department of Health and Social Care. Patient consent for publication Not applicable. CONCLUDING REMARKS yp y y 4 Pennamen P, Tingaud-Sequeira A, Gazova I, et al. Dopachrome tautomerase variants in patients with oculocutaneous albinism. Genet Med 2021;23:479–87. We describe a framework for analysing genetic test results from individuals suspected of having albinism. The impor- tance of incorporating selected common genetic variants 5 Lasseaux E, Plaisant C, Michaud V, et al. Molecular characterization of a series of 99 index patients with albinism. Pigment Cell Melanoma Res 2018;31:466–74. Sergouniotis PI, et al. J Med Genet 2023;60:1245–1249. doi:10.1136/jmg-2022-109088 1248 Vision science 14 Mantere T, Kersten S, Hoischen A. Long-read sequencing emerging in medical genetics. Front Genet 2019;10:426. 6 Niemi MEK, Martin HC, Rice DL, et al. Common genetic variants contribute to risk of rare severe neurodevelopmental disorders. Nature 2018;562:268–71. g 15 Lenassi E, Clayton-Smith J, Douzgou S, et al. Clinical utility of genetic testing in 201 preschool children with inherited eye disorders. Genet Med 2020;22:745–51. p 7 Won H, Huguet G, Jacquemont S. Rare and common autism risk variants converge across 16p. Nat Genet 2022;54:1587–8. p 8 Michaud V, Lasseaux E, Green DJ, et al. The contribution of common regulatory and protein-coding TYR variants to the genetic architecture of albinism. Nat Commun 2022;13:3939. p 8 Michaud V, Lasseaux E, Green DJ, et al. The contribution of common regulatory and protein-coding TYR variants to the genetic architecture of albinism. Nat Commun 2022;13:3939. 16 Liu B, Calton MA, Abell NS, et al. Genetic analyses of human fetal retinal pigment epithelium gene expression suggest ocular disease mechanisms. Commun Biol 2019;2:186. 9 Richards S, Aziz N, Bale S, et al. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genetics in Medicine 2015;17:405–24. 17 Reinisalo M, Putula J, Mannermaa E, et al. Regulation of the human tyrosinase gene in retinal pigment epithelium cells: The significance of transcription factor Orthodenticle Homeobox 2 and its polymorphic binding site. Mol Vis 2012;18:38–54. http://jmg.bmj.com/ published as 10.1136/jmg-2022-109088 on 17 July 2023. Downloaded from 10 Houge G, Laner A, Cirak S, et al. Stepwise ABC system for classification of any type of genetic variant. Eur J Hum Genet 2022;30:150–9. 18 Jagirdar K, Smit DJ, Ainger SA, et al. Molecular analysis of common polymorphisms within the human tyrosinase locus and genetic association with pigmentation traits. Pigment Cell Melanoma Res 2014;27:552–64. Sergouniotis PI, et al. J Med Genet 2023;60:1245–1249. doi:10.1136/jmg-2022-109088 CONCLUDING REMARKS 11 King RA, Townsend D, Oetting W, et al. Temperature-sensitive tyrosinase associated with peripheral pigmentation in oculocutaneous albinism. J Clin Invest 1991;87:1046–53. 19 Tripathi RK, Giebel LB, Strunk KM, et al. A polymorphism of the human tyrosinase gene is associated with temperature-sensitive enzymatic activity. Gene Expr 1991;1:103–10. g 12 Monfermé S, Lasseaux E, Duncombe-Poulet C, et al. Mild form of oculocutaneous albinism type 1: Phenotypic analysis of compound heterozygous patients with the R402Q variant of the TYR gene. Br J Ophthalmol 2019;103:1239–47. 20 Toyofuku K, Wada I, Valencia JC, et al. Oculocutaneous albinism types 1 and 3 are ER retention diseases: Mutation of tyrosinase or TYRP1 can affect the processing of both mutant and wild-type proteins. FASEB J 2001;15:2149–61. 20 Toyofuku K, Wada I, Valencia JC, et al. Oculocutaneous albinism types 1 and 3 are ER retention diseases: Mutation of tyrosinase or TYRP1 can affect the processing of both mutant and wild-type proteins. FASEB J 2001;15:2149–61. g p 13 Lin S, Sanchez-Bretaño A, Leslie JS, et al. Evidence that the Ser192Tyr/Arg402Gln in cis tyrosinase gene haplotype is a disease-causing allele in oculocutaneous albinism type 1B (OCA1B). NPJ Genom Med 2022;7:2. Sergouniotis PI, et al. J Med Genet 2023;60:1245–1249. doi:10.1136/jmg-2022-109088 1249
https://openalex.org/W3217704168	https://www.engiscience.com/index.php/josse/article/download/josse2021121/pdf	English	null	Scheduling and Synchronization Algorithms in Operating System: A Survey	Journal of studies in science and engineering	2,021	cc-by	7,563	Review Article An overview of each technique with the main algorithms have been described in detail with the advantages and the issues of each algorithm. Furthermore, this paper has dug deep into the real-time operating system scheduling issues, which is the current trend in operating system researches. Keywords CPU Scheduling Multiprocessor Round-Robin Scheduling Priority Scheduling Real-time Copyright: © 2021 Mohammed Y. Shakor. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY 4.0) license . i Copyright: © 2021 Mohammed Y. Shakor. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY 4.0) license . Copyright: © 2021 Mohammed Y. Shakor. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY 4.0) license . Journal of Studies in Science and Engineering. 2021, 1(2), 1-16. https://doi.org/10.53898/josse2021121 Journal of Studies in Science and Engineering Journal of Studies in Science and Engineering Review Article Review Article Review Article Scheduling and Synchronization Algorithms in Operating System: A Survey Article Info Abstract Article History Received Sep 20, 2021 Revised Oct 23, 2021 Accepted Nov 08, 2021 An operating system is software that is designed to manage computer hardware and software resources. However, this management requires applying an ample number of techniques and algorithms which are called synchronization and scheduling. The scheduling algorithms are used to arrange the way that the CPU is assigned to the processes, while synchronization is utilized to indicate how to work with multi-processes at the same time. Therefore, they are related to each other. CPU scheduling is a vital phenomenon of an operating system. At present, numerous CPU scheduling algorithms exist as First Come First Serve )FCFS(, Shortest Job First (SJF), Shortest Remaining Time First (SRTF), Priority Scheduling, and Round Robin (RR). In this paper, a survey of the current synchronization and scheduling algorithms have been presented. An overview of each technique with the main algorithms have been described in detail with the advantages and the issues of each algorithm. Furthermore, this paper has dug deep into the real-time operating system scheduling issues, which is the current trend in operating system researches. Keywords CPU Scheduling Multiprocessor Round-Robin Scheduling Priority Scheduling Real-time Copyright: © 2021 Mohammed Y. Shakor. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY 4.0) license . 1 I t d ti Article Info Abstract Article History Received Sep 20, 2021 Revised Oct 23, 2021 Accepted Nov 08, 2021 An operating system is software that is designed to manage computer hardware and software resources. However, this management requires applying an ample number of techniques and algorithms which are called synchronization and scheduling. The scheduling algorithms are used to arrange the way that the CPU is assigned to the processes, while synchronization is utilized to indicate how to work with multi-processes at the same time. Therefore, they are related to each other. CPU scheduling is a vital phenomenon of an operating system. At present, numerous CPU scheduling algorithms exist as First Come First Serve )FCFS(, Shortest Job First (SJF), Shortest Remaining Time First (SRTF), Priority Scheduling, and Round Robin (RR). In this paper, a survey of the current synchronization and scheduling algorithms have been presented. 1. Introduction Back in the old days of batch systems with input in the form of card images on a magnetic tape, the scheduling algorithm was simple: just run the next job on the tape. With timesharing systems, the schedul- ing algorithm became more complex, because there were generally multiple users waiting for service. There may be one or more batch streams as well (e.g., at an insurance company, for processing claims). On a personal computer, you might think there would be only one active process. After all, a user entering a document on a word processor is unlikely to be simultaneously compiling a program in the background. However, there are often background jobs, such as electronic mail daemons sending or receiving e-mail. You might also think that computers have gotten so much faster over the years that the CPU is rarely a scarce resource anymore. However, new applications tend to demand more resources. Processing digital photographs or watching real-time videos are examples. The problem of scheduling is concerned with searching for optimal (or near-optimal) schedules sub- ject to several constraints. A variety of approaches have been developed to solve the problem of scheduling. urnal of Studies in Science and Engineering. 2021, 1(2), 1-16. https://doi.org/10.53898/josse2021121 https://engiscience.com/index.php/josse M. Shakor 2021 2 However, many of these approaches are often impractical in dynamic real-world environments where there are complex constraints and a variety of unexpected disruptions. In most real-world environments, scheduling is an ongoing reactive process where the presence of real-time information continually forces reconsideration and revision of pre-established schedules. Scheduling research has largely ignored this problem, focusing instead on the optimization of static schedules. This paper outlines the limitations of static approaches to scheduling in the presence of real-time information and presents many issues that have come up in recent years on dynamic scheduling. In this paper, an overview of both synchronization and scheduling techniques in OS are going to be introduced, addressing the advantages and disadvantages of using algorithms in both techniques and the issues that call to use synchronization and scheduling. Journal of Studies in Science and Engineering. 2021, 1(2), 1-16. https://doi.org/10.53898/josse2021121 https://engiscience.com/index.php/josse 2. Scheduling Criteria Various CPU scheduling algorithms have different properties, and the choice of a particular algorithm may favour one class of processes over another [1]. For selection of an algorithm for a particular situation, we must consider the properties of various algorithms. The scheduling criteria include the following [2]: • Context Switch: A context switch is a process of storing and restoring context (state) of a preempted process, so that execution can be resumed from the same point at a later time. Context switching is usually computationally intensive, leading to wastage of time and memory, which in turn increases the overhead of the scheduler, so the design of the operating system is to optimize only these switches. • Throughput: Throughput is defined as a number of processes completed per unit time. Throughput is slow in round-robin scheduling implementation. Context switching and throughput are inversely pro- portional to each other. • CPU Utilization: This is a measure of how much busy the CPU is. Usually, the goal is to maximize CPU utilization. • CPU Utilization: This is a measure of how much busy the CPU is. Usually, the goal is to maximize CPU utilization. • Turnaround Time: Turnaround time refers to the total time which is spent to complete the process and is how long it takes the time to execute that process. The time interval from the time of submission of a process to the time of completion is the turnaround time. Total turnaround time is the sum of the periods spent waiting to get into memory, waiting time in the ready queue, execution time on the CPU and doing I/O. • Waiting Time: Waiting time is the total time a process has been waiting in the ready queue. The CPU scheduling algorithm does not affect the amount of time during which a process executes or does input-output; it affects only the amount of time that a process spends waiting in a ready queue. • Response Time: In an interactive system, turnaround time may not be the best measure. Often, a process can produce some output fairly early and can continue computing new results while previous Journal of Studies in Science and Engineering. 2021, 1(2), 1-16. https://doi.org/10.53898/josse2021121 https://engiscience.com/index.php/josse https://engiscience.com/index.php/josse Scheduling and Synchronization Algorithms in Operating System: A Survey 3 results are being produced to the user. 2. Scheduling Criteria Thus, response time is the time from the submission of a request until the first response is produced that means time when the task is submitted until the first response is received. So, the response time should be low for best scheduling. Journal of Studies in Science and Engineering. 2021, 1(2), 1-16. https://doi.org/10.53898/josse2021121 https://engiscience.com/index.php/josse 3.1 First-Come, First-Served Scheduling First-Come-First-Served (FCFS) is the simplest scheduling algorithm, it simply queues processes in the order that they arrive in the ready queue. Processes are dispatched according to their arrival time on the ready queue. Being a non-preemptive discipline, once a process has a CPU, it runs to completion. The FCFS scheduling is fair in the formal sense or human sense of fairness but it is unfair in the sense that long jobs make short jobs wait and unimportant jobs make important jobs wait. 3. Scheduling Algorithm Maximize throughput: A scheduling algorithm should be capable of servicing the maximum number of processes per unit of time. 2. Degrade gracefully under heavy load. 3. Avoid indefinite blocking or starvation: A process should not wait for an unbounded time before or while processing service. 4. Enforcement of priorities: if the system assigns priorities to processes, the scheduling mechanism should favor the higher-priority processes. 5. Minimize overhead: Overhead causes wastage of resources. But when we use system resources effectively, then overall system performance improves greatly. 6. Achieve balance between response and utilization: The scheduling mechanism should keep the re- sources of the system busy. 7. Favor processes exhibit desirable behavior. 3. Scheduling Algorithm Scheduling is characterized as the portion of assets to employments after some time. It is a basic leadership process to improve at least one destination. The goals can be the minimization of the finishing time of occupations, mean stream time, the delay of employments, preparing cost, and so on [1]. Scheduling assumes a vital part in numerous assembling and creation frameworks. Planning issues, which are worried about hunting down ideal (or close ideal) prescient timetables subject to various limita- tions, are for the most part NP-hard. Up until this point, studies have principally been centred around finding ideal (or close ideal) answers for static models as for different measures, i.e. most brief aggregate preparing time, negligible creation cost, and so on. These methodologies for the most part have utilized the verifiable supposition of static conditions with no sort of disappointments. CPU scheduling is a key concept in com- puter multitasking, multiprocessing operating systems and real‐time operating system designs. Scheduling refers to the way processes are assigned to run on the available CPUs, since there are typically many more processes running than there are available CPUs [1]. CPU scheduling deals with the problem of deciding which of the processes in the ready queue is to be allocated the CPU [2]. By switching the CPU among processes, the operating system can make the computer more productive. A multiprogramming operating system allows more than one process to be loaded into the executable memory at a time and for the loaded processes to share the CPU using time multiplexing. Figure 1 shows the main principles of CPU scheduling with the states in the CPU. Figure 1. Basic CPU scheduler Figure 1. Basic CPU scheduler The method by which threads, processes or data flows are given access to system resources (e.g. processor time, communications bandwidth) is called a scheduling algorithm. The need for a scheduling Journal of Studies in Science and Engineering. 2021, 1(2), 1-16. https://doi.org/10.53898/josse2021121 https://engiscience.com/index.php/josse M. Shakor 2021 4 algorithm arises from the requirement for most modern systems to perform multitasking (execute more than one process at a time) and multiplexing (transmit multiple flows simultaneously). A system designer must consider a variety of factors in designing a scheduling algorithm, such as the type of systems used and what are the user's needs. Depending on the system, the user and designer might expect the scheduler to: 1. Journal of Studies in Science and Engineering. 2021, 1(2), 1-16. https://doi.org/10.53898/josse2021121 https://engiscience.com/index.php/josse complete. Also, it undergoes a starvation issue especially in a busy system with many small processes being run. complete. Also, it undergoes a starvation issue especially in a busy system with many small processes being run. 3.3 Priority Scheduling The operating system assigns a fixed priority to every process, and the scheduler arranges the pro- cesses in the ready queue in order of their priority. Lower priority processes get interrupted by incoming higher priority processes. Overhead is not minimal, nor is it significant in this case. Waiting time and response time depend on the priority of the process. Higher priority processes have smaller waiting and response times. Deadlines can be easily met by giving higher priority to the earlier deadline processes. Disadvantage: Starvation of lower priority processes is possible if a large no of higher priority processes keeps arriving continuously[3]. Journal of Studies in Science and Engineering. 2021, 1(2), 1-16. https://doi.org/10.53898/josse2021121 3.2 Shortest-Job-First Scheduling Shortest Job First (SJF) is the strategy of arranging processes with the least estimated processing time remaining to be next in the queue. It works under the two schemes (preemptive and non-preemptive). It’s probably optimal since it minimizes the average turnaround time and the average waiting time. The main problem with this discipline is the necessity of previous knowledge about the time required for a process to urnal of Studies in Science and Engineering. 2021, 1(2), 1-16. https://doi.org/10.53898/josse2021121 https://engiscience.com/index.php/josse https://engiscience.com/index.php/josse Scheduling and Synchronization Algorithms in Operating System: A Survey 5 complete. Also, it undergoes a starvation issue especially in a busy system with many small processes being run. 3.4 Round-Robin Scheduling Round Robin (RR) is one of the oldest, simplest and fairest and most widely used scheduling algo- rithms, designed especially for time-sharing systems [4]. It’s designed to give a better response but the worst turnaround and waiting time due to the fixed time quantum concept. The scheduler assigns a fixed time unit (quantum) per process usually 10-100 milliseconds, and cycles through them. RR is similar to FCFS except that preemption is added to switch between processes. Figure 2. Round-Robin algorithm example Figure 2. Round-Robin algorithm example The RR planning calculation is given by the following advances [4]: The RR planning calculation is given by the following advances [4]: The RR planning calculation is given by the following advances [4]: 1. The scheduler keeps up a line of prepared procedures and a rundown of shut and swapped out proce- dures. 1. The scheduler keeps up a line of prepared procedures and a rundown of shut and swapped out proce- dures. Journal of Studies in Science and Engineering. 2021, 1(2), 1-16. https://doi.org/10.53898/josse2021121 https://engiscience.com/index.php/josse M. Shakor 2021 6 2. The Process Control Block of a recently made process is added to the end of a prepared line. The Process Control Block of the ending process is expelled from the booking information structures. 2. The Process Control Block of a recently made process is added to the end of a prepared line. The Process Control Block of the ending process is expelled from the booking information structures. 3. The scheduler dependably chooses the Process Control Block from the leader of the prepared line. This is a drawback since all procedures are fundamentally given a similar need. Round robin additionally supports the procedure with short CPU bursts and punishes long ones. 3. The scheduler dependably chooses the Process Control Block from the leader of the prepared line. This is a drawback since all procedures are fundamentally given a similar need. Round robin additionally supports the procedure with short CPU bursts and punishes long ones. 4. When a running procedure completes its opportunity cut, it is moved to the end of a prepared line. A period cut is a measure of time that each procedure spends on the processor per emphasis of the Round Robin calculation. All procedures are executed in first start things out serve way however are seized after a period cut. Journal of Studies in Science and Engineering. 2021, 1(2), 1-16. https://doi.org/10.53898/josse2021121 3.5 Multilevel Queue Scheduling In the circumstance in which the procedure is partitioned into various gatherings, multi-level line planning is utilized. The attributes of multi-level planning are as per the following: Based on the sorts, the procedures are isolated into various lines. Procedures are doled out to one line for all time. The booking calculation in each line is extraordinary. For instance, as appeared in the figure beneath, intelligent process and clump employment may use the Round Robin planning technique and FCFS strategy separately. In the circumstance in which the procedure is partitioned into various gatherings, multi-level line planning is utilized. The attributes of multi-level planning are as per the following: Based on the sorts, the procedures are isolated into various lines. Procedures are doled out to one line for all time. The booking calculation in each line is extraordinary. For instance, as appeared in the figure beneath, intelligent process and clump employment may use the Round Robin planning technique and FCFS strategy separately. Additionally, the line must be planned and for the most part, it has settled need. A typical division between frontal area procedures and foundation forms is made. These two kinds of procedures may contrast in their reaction times; thusly, they may require distinctive booking. The frontal area procedures may have need over foundation procedures and this need might be characterized remotely. The bunch line must be executed when the line for framework executes and the intelligent procedures are sat without moving or exhausting. If the bunch procedure is still in process and the intelligent procedure enters the prepared line, the cluster will be avoided. 3.4 Round-Robin Scheduling https://doi.org/10.53898/josse2021121 https://engiscience.com/index.php/josse https://engiscience.com/index.php/josse Scheduling and Synchronization Algorithms in Operating System: A Survey 7 • Low throughput: Throughput is defined as the number of processes completed per time unit. If round robin is implemented in soft real-time systems throughput will be low which leads to severe degradation of system performance. If the number of context switches is low then the throughput will be high. Context switch and throughput are inversely proportional to each other. • Low throughput: Throughput is defined as the number of processes completed per time unit. If round robin is implemented in soft real-time systems throughput will be low which leads to severe degradation of system performance. If the number of context switches is low then the throughput will be high. Context switch and throughput are inversely proportional to each other. 3.4 Round-Robin Scheduling The procedure will either complete in the time cut given or the procedure will come back to the tail of the prepared line and come back to the processor at a later time. 5. The occasion handler plays out the accompanying activities: a. When a procedure influences a contribution to yield ask for or swapped out, its Process Control Block is expelled from prepared line to blocked/swapped out rundown. a. When a procedure influences a contribution to yield ask for or swapped out, its Process Control Block is expelled from prepared line to blocked/swapped out rundown. b. When input-yield activity anticipated by a procedure completes or process is swapped in its Process Control Block is expelled from blocked/swapped rundown to the finish of the prepared line. b. When input-yield activity anticipated by a procedure completes or process is swapped in its Process b. When input-yield activity anticipated by a procedure completes or process is swapped in its Process Control Block is expelled from blocked/swapped rundown to the finish of the prepared line. Control Block is expelled from blocked/swapped rundown to the finish of the prepared line. There are some disadvantages of the Round Robin CPU scheduling algorithm for the operating system which are as follows: There are some disadvantages of the Round Robin CPU scheduling algorithm for the operating system which are as follows: • Larger waiting time and response time: In the Round Robin architecture the time which a process spends in the ready queue waiting for the processor to get executed is known as waiting time and the time when the process takes to complete its job and exit from the task is called as turnaround time. Larger waiting and response time are a drawback in the Round Robin architecture as it leads to degradation of system performance. • Context Switches: When the time slice of the task ends and the task is still executing on the proces- sor the scheduler forcibly preempts the tasks on the processor and stores the task context in stack or registers and allocates the processor to the next task in the ready queue. This action which is per- formed by the scheduler is called a context switch. Context switch leads to the wastage of time, memory and leads to scheduler overhead. Journal of Studies in Science and Engineering. 2021, 1(2), 1-16. Journal of Studies in Science and Engineering. 2021, 1(2), 1-16. https://doi.org/10.53898/josse2021121 3.7 Non-preemptive scheduling algorithm Non-preemptive or also known as cooperative scheduling is the first scheme where once a process has control of the CPU no other processes can preemptively take the CPU away. The process retains the CPU until either it terminates or enters the waiting state. Two algorithms can be used for non-preemptive scheduling. 3.6 Multilevel Feedback Queue Scheduling MLFQ utilizing Three lines: In MLFQ (Multi-level criticism line) planning, the line is separated into three sections where two lines have Round Robin booking method and the staying one has FCFS booking system [5]. Every one of the procedures is arranged in the first line as indicated by their burst time and after that, they are permitted to execute for a particular time. At the point when the procedures finish their un- derlying execution, they are arranged in the second line as per their outstanding burst time. After the exe- cution, the procedures are moved to the third line where they are arranged to keep running with the FCFS booking method. This calculation limits the holding up time and turnaround time however CPU needs to hold up to assemble a line of the considerable number of procedures. It is the fundamental driver that re- stricts the best usage of assets. https://engiscience.com/index.php/josse https://engiscience.com/index.php/josse M. Shakor 2021 8 MLFQ utilizing Five lines: In this procedure, the procedures are planned for five distinct lines. Start- ing need isn't relegated to the procedure yet they are booked utilizing Round Robin planning with a reason- able time quantum esteem when the procedure is started. The procedures are booked and permitted to keep running in a line as per their burst time [6]. At last, either the procedures are finished or they are arranged to keep running into the second line to run again with the rest of the burst time and holding-up time. This booking is finished utilizing the Round Robin CPU planning system which likewise doles out an appropri- ate time quantum esteem. Holding up time and remaining CPU burst time and turnaround time are the principal parameters that are ascertained and refreshed in each progression. Toward the finish of the second line, either the procedures get finished or they are additionally arranged to the next line till every one of the procedures gets executed. A few procedures need to wait for a long time for execution. This planning cal- culation executes every one of the procedures in parallel. Along these lines, this calculation expels the starvation issue of various procedures. For this situation, the quantity of switches is more a direct result of the capacity and estimations of burst time and holding-up time of each line. Journal of Studies in Science and Engineering. 2021, 1(2), 1-16. https://doi.org/10.53898/josse2021121 4. Multiple-Processor Scheduling When a computer system contains multiple processors, a few new issues arise. Multiprocessor systems can be categorized into the following: loosely coupled or distributed. The latter consists of a collection of relatively autonomous systems connected with an interconnection network. Each of them has its memory and I/O Channels [7]. A system with functionality specialized processors, or servers, such as an I/O, network, graphics, or a math coprocessor, works in an environment controlled by a general-purpose, master processor, to provide specific services. A tightly coupled multiprocessing system consists of processors that share a common memory and are under the control of an operating system. The presently popular multi- core architecture falls into this category. urnal of Studies in Science and Engineering. 2021, 1(2), 1-16. https://doi.org/10.53898/josse2021121 https://engiscience.com/index.php/josse https://engiscience.com/index.php/josse Scheduling and Synchronization Algorithms in Operating System: A Survey 9 One way to characterize and compare multiprocessor systems is to consider their synchronization granularity, namely, the frequency of synchronization between processors in a system. We can thus categorize parallelism in terms of their granularity degree; with independent parallelism, there is no explicit synchronization among processes. Each process in the system represents a separate, independent application, or job. For example, in a time-sharing system such as turning, each user performs a particular application, such as c programming, system services, database related stuff, etc. The multiprocessor system thus provides the same service as a multi-programmed uniprocessor system, but with less response time from the perspective of a user. With coarse and very coarse-grained parallelism, there is minimum synchronization among processes. This kind of situation can be easily handled as a set of concurrent processes running on a multi-programmed uniprocessor system and can be supported on a multiprocessor with little chance of the associated software. As an example, a program has been developed which takes in specifications of files that need recompilation and decides which of these compilations can be done simultaneously. It is reported that the actual speedup is more than expected since some of the compiled codes can be shared. In these situations, the linear speedup is certainly what we can expect the most. Because the threads coming from a single process interact among themselves so frequently, scheduling decisions concerning one thread may have some impact on other threads belonging to the same process. Journal of Studies in Science and Engineering. 2021, 1(2), 1-16. https://doi.org/10.53898/josse2021121 https://engiscience.com/index.php/josse 4. Multiple-Processor Scheduling When dealing with a multiprocessor system, besides the dispatching policies, we have to talk about a few other issues, including how to assign processors to processes, now that we have more to give away, how to make use of multiprogramming on individual processors. Assume that we have a fair and uniform environment, then the simplest approach of processor assignment is to treat all the processors as a pool. If a processor is permanently assigned to a process throughout its life, we should associate a short-term queue with each processor. This will lead to smaller overhead, but an uneven workload for processors. An alternative is to use a common queue to serve all the processes. Thus, a process can run on different processors at different times. Regarding the actual assignment, at least two approaches can be followed: With a master/slave approach, key kernel functions, including the scheduler of the operating system, always run on a “master” processor, while the rest of the processors are used to run user processes. When a slave process needs some urnal of Studies in Science and Engineering. 2021, 1(2), 1-16. https://doi.org/10.53898/josse2021121 https://engiscience.com/index.php/josse https://engiscience.com/index.php/josse M. Shakor 2021 10 service, it simply sends a request to the master processor and waits for its response. This approach is very simple and does not need a conflicting resolution mechanism. But the master can become a bottleneck, and it can even bring down the whole system when it fails. In a peer structure, the OS can execute on any processor, and each of them does its scheduling among the available processes. This certainly makes the situation messier, since the OS must ensure that two processors will not choose the same process and no process is starved, i.e., never gets chosen. Also, competition among processes for various resources must be resolved. There is plenty of room between these two extremes. For example, a subset of processors, instead of just one processor, can be selected to run the kernel functions, including the dispatcher policies. Journal of Studies in Science and Engineering. 2021, 1(2), 1-16. https://doi.org/10.53898/josse2021121 4.1 Multicore Processors Driven by a performance-hungry market, microprocessors have always been designed keeping performance and cost in mind. Gordon Moore, the founder of Intel Corporation, predicted that the number of transistors on a chip will double once every 18 months to meet this ever-growing demand which is popularly known as Moore’s Law in the semiconductor industry. Advanced chip fabrication technology alongside integrated circuit processing technology offers increasing integration density which has made it possible to integrate one billion transistors on a chip to improve performance. However, the performance increase by micro-architecture governed by Pollack’s rule is roughly proportional to the square root of the increase in complexity. This would mean that doubling the logic on a processor core would only improve the performance by 40%. With advanced chip fabrication techniques comes along another major bottleneck: power dissipation issue. Studies have shown that transistor leakage current increases as the chip size shrinks further and further which increases static power dissipation to large values. One alternative means of improving performance is to increase the frequency of operation which enables faster execution of programs. However, the frequency is again limited to 4GHz currently as any increase beyond this frequency increases power dissipation again. “Battery life and system cost constraints drive the design team to consider power over performance in such a scenario” [8]. Power consumption has increased to such high levels that traditional air-cooled microprocessor server boxes may require budgets for liquid cooling or refrigeration hardware. Designers eventually hit what is referred to as the power wall, the limit on the amount of power a microprocessor could dissipate. https://engiscience.com/index.php/josse https://engiscience.com/index.php/josse Scheduling and Synchronization Algorithms in Operating System: A Survey 11 “A Multi-core processor is typically a single processor which contains several cores on a chip” [9]. The cores as shown in Figure 3 are functional units made up of computation units and caches. These multiple cores on a single chip combine to replicate the performance of a single faster processor. The individual scores on a multi-core processor don’t necessarily run as fast as the highest performing single- core processors, but they improve overall performance by handling more tasks in parallel. The performance boost can be seen by understanding how single-core and multi-core processors execute programs. Single- core processors running multiple programs would assign time slices to work on one program and then assign different time slices for the remaining programs. 4.1 Multicore Processors If one of the processes is taking a long time to complete then all the rest of the processes start lagging. However, In the case of multi-core processors, if you have multiple tasks that can be run in parallel at the same time, each of them will be executed by a separate core in parallel thus boosting the performance. Figure 3. Multi-core processor Figure 3. Multi-core processor Journal of Studies in Science and Engineering. 2021, 1(2), 1-16. https://doi.org/10.53898/josse2021121 5. Real-Time CPU Scheduling Real-time systems are characterized as those systems in which the accuracy of the system depends on the coherent consequence of calculation, as well as on the time at which the outcomes are created. Cases of this kind of real-time system are the charge and control system, process control system, flight real-time system, the Space Shuttle aeronautics system, future system, for example, the space station, space-based safeguard system, for example, SDI, and expansive order and control system. A dominant part of the current systems expects that quite a bit of this learning is accessible from earlier and subsequently depend on static plans which add to their high cost and resoluteness. The cutting-edge hard ongoing system must be intended to be dynamic, unsurprising, and adaptable. https://engiscience.com/index.php/josse https://engiscience.com/index.php/josse M. Shakor 2021 12 A real-time operating system must be able to perform integrated CPU scheduling and resource allocation so that collections of cooperating tasks can obtain the resources they need, at the right time, to meet timing constraints. In addition to proper scheduling algorithms, predictability requires bounded operating system primitives. Scheduling involves the allocation of resources and time to tasks in such a way that certain performance requirements are met [10]. Scheduling has been perhaps the most widely researched topic within real-time systems. This is due to the belief that the basic problem in real-time systems is to make sure that tasks meet their time constraints. Consistency is one of the essential issues of a real-time system. Schedulability investigation or practicality checking of the errands of a real-time system must be done to anticipate whether the assignments will meet their planning imperatives. A few planning ideal models develop, contingent upon a) regardless of whether a system performs schedulability examination, b) on the off chance that it does, whether it is done statically or powerfully, and c) whether the consequence of the investigation itself creates a calendar or plan as indicated by which errands are dispatched at run-time. Because of this, we can distinguish the accompanying classes of calculations: • Static table-driven approaches: These perform static schedulable investigation and the subsequent calendar (or table, as it is typically called) is utilized at runtime to choose when an errand must start execution [11]. • Static priority-driven preemptive approaches: These perform static schedulable investigation yet not at all like in the past approach, no express calendar is built. Journal of Studies in Science and Engineering. 2021, 1(2), 1-16. https://doi.org/10.53898/josse2021121 Journal of Studies in Science and Engineering. 2021, 1(2), 1-16. https://doi.org/10.53898/josse2021121 https://engiscience.com/index.php/josse 5.1 Real-time Operating System Real-time operating systems are an integral part of real-time systems. Not surprisingly, the four main functional areas that they support are process management and synchronization, memory management, inter-process communication, and I/O. However, how they support these areas differs from conventional operating systems as will be discussed in this section. In particular, real-time operating systems stress predictability and include features to support real-time constraints. Three general categories of real-time operating systems exist: small, proprietary kernels (commercially available as well as homegrown kernels), real-time extensions to commercial timesharing operating systems such as UNIX, and research kernels. these three main categories of real-time operating systems [13]. 5. Real-Time CPU Scheduling At runtime, errands are executed "most elevated need first." [12]. • Dynamic planning-based approaches: Unlike the past two methodologies, attainability is checked at runtime, i.e., a powerfully arriving assignment is acknowledged for execution only if it is found fea- sible. (Such a task is said to be guaranteed to meet its time constraints.) One of the results of the feasibility analysis is a schedule or plan that is used to decide when a task can begin execution. • Dynamic best effort approaches: Here no feasibility checking is done. The system tries to do its best to meet deadlines. But since no guarantees are provided, a task may be aborted during its execution [13]. https://engiscience.com/index.php/josse https://engiscience.com/index.php/josse Scheduling and Synchronization Algorithms in Operating System: A Survey 13 5.1.1 Small, Fast, Proprietary Kernels The little, quick, exclusive portions come in two assortments: homegrown and business offerings' [14]. The two assortments are frequently utilized for little inserted frameworks when quick and exceptionally unsurprising execution must be ensured. The homegrown bits are typically very specific to the application. The cost of interestingly creating and keeping up a homegrown piece, and in addition the expanding nature of the business offerings is fundamentally decreasing the act of producing homegrown portions. For the two assortments of exclusive portions, to accomplish speed and consistency, the pieces are stripped down and advanced variants of time-sharing working frameworks. Journal of Studies in Science and Engineering. 2021, 1(2), 1-16. https://doi.org/10.53898/josse2021121 https://engiscience.com/index.php/josse 5.1.3 Research Operating System While many real-time applications will continue to be constructed with proprietary real-time kernels and with extensions to commercial time-sharing operating systems, as discussed above, significant problems still exist [15]. In particular, the proprietary kernels have difficulty when scaling to large applications, and the time-sharing extensions emphasize speed rather than predictability, thereby perpetuating the myth that real-time computing is fast computing. 5.1.2 Real-Time Extensions to Commercial Operating Systems A second way to deal with a real-time operating system is the augmentation of business items, e.g., stretching out UNIX to RT-UNIX, or POSIX to RT-POSIX, or MACH to RT-MACH, or CHORUS to a constant form [14]. The real-time version of a commercial operating system is, for the most part, slower and less unsurprising than the restrictive pieces, yet have more prominent usefulness and better programming improvement situations imperative contemplations in numerous applications. Another critical favorable position is that they depend on an arrangement of well-known interfaces (norms) that encourage transportability. For UNIX, since numerous varieties of UNIX have developed another model’s exertion, called POSIX which has characterized a typical arrangement of client level interfaces for operating systems. Specifically, the POSIX P. 1003.4 subcommittee is characterizing measures for a real- https://engiscience.com/index.php/josse https://engiscience.com/index.php/josse M. Shakor 2021 14 time operating system. To date, the exertion has concentrated on eleven essential continuous related capacities: clocks, need planning, shared memory, ongoing documents, semaphores, inter-process correspondence, offbeat occasion warning, process memory locking, nonconcurrent I/O, synchronous I/O, and strings. Journal of Studies in Science and Engineering. 2021, 1(2), 1-16. https://doi.org/10.53898/josse2021121 https://engiscience.com/index.php/josse Journal of Studies in Science and Engineering. 2021, 1(2), 1-16. https://doi.org/10.53898/josse2021121 https://engiscience.com/i 6.1 Critical Section Problem Mutual exclusion is a fundamental process synchronization problem in concurrent systems, and it has been studied extensively in the last half-century [17]. Generalizations of mutual exclusion have been studied extensively as well. Such generalizations include k-mutual exclusion, mutual inclusion, and -mutual inclusion. The number of processes that are in the critical section simultaneously is at most k by k-mutual exclusion, while that is at least by -mutual inclusion. Unfortunately, these generalized problems have been studied individually [18]. 6. Synchronization In some operating systems, processes that are working together may share some common storage that each one can read and write. The shared storage may be in the main memory (possibly in a kernel data structure) or it may be a shared file; the location of the shared memory does not change the nature of the communication or the problems that arise [16]. To see how interprocess communication works in practice, let us consider a simple but common example: a print spooler. When a process wants to print a file, it enters the file name in a special spooler directory. Another procedure, the printer daemon, intermittently verifies whether there are any documents to be printed, and if there are, it prints them and after that expels their names from the index. Envision that our spooler index has countless, numbered 0, 1, 2 every one fit for holding a record name. Additionally, envision that there are two shared factors: out, which focuses on the following document to be printed, and in, which focuses on the following free space in the catalogue. These two factors may well be kept on a two-word record accessible to all procedures. At a specific moment, spaces 0 to 3 are purged (the records have just been printed) and spaces 4 to 6 are full (with the names of documents lined for printing). Pretty much at the same time, forms An and B choose they need to line a document for printing. The way to avert the inconvenience of race condition here and in numerous different circumstances including shared memory shared documents, and shared everything else is to discover some approach to https://engiscience.com/index.php/josse https://engiscience.com/index.php/josse Scheduling and Synchronization Algorithms in Operating System: A Survey 15 preclude more than one process from perusing and composing the mutual information in the meantime. Put as such, what we require is common rejection, that is, some method for ensuring that on the off chance that one procedure is utilizing a shared variable or record, alternate procedures will be prohibited from doing likewise thing. The trouble above happened because procedure B began utilizing one of the shared factors previously process A was done with it. The decision of proper crude activities for accomplishing shared rejection is a noteworthy outline issue in any working framework and a subject that we will inspect in extraordinary detail in the accompanying segments. 6. Synchronization preclude more than one process from perusing and composing the mutual information in the meantime. Put as such, what we require is common rejection, that is, some method for ensuring that on the off chance that one procedure is utilizing a shared variable or record, alternate procedures will be prohibited from doing likewise thing. The trouble above happened because procedure B began utilizing one of the shared factors previously process A was done with it. The decision of proper crude activities for accomplishing shared rejection is a noteworthy outline issue in any working framework and a subject that we will inspect in extraordinary detail in the accompanying segments. The issue of maintaining a strategic distance from race conditions can likewise be planned conceptually. Some portion of the time, a procedure is occupied with doing inside calculations and other things that don't prompt race conditions. In any case, some of the time a procedure needs to get to shared memory or documents, or do other basic things that can prompt races. That piece of the program where the mutual memory is gotten to is known as the basic locale or basic segment. On the off chance that we could orchestrate matters to such an extent that no two procedures were ever in their basic areas in the meantime, we could keep away from races. Journal of Studies in Science and Engineering. 2021, 1(2), 1-16. https://doi.org/10.53898/josse2021121 References [1] M. L. Pinedo, Scheduling: theory, algorithms, and systems. Springer, 2016. [2] K. Ramamritham and J. A. Stankovic, “Scheduling algorithms and operating systems support for real-time systems,” Proc. IEEE, vol. 82, no. 1, pp. 55–67, 1994. [3] B. Andersson, S. Baruah, and J. Jonsson, “Static-priority scheduling on multiprocessors,” in Real-Time Systems Symposium, 2001.(RTSS 2001). Proceedings. 22nd IEEE, 2001, pp. 193–202. [4] A. Singh, P. Goyal, and S. Batra, “An optimized round robin scheduling algorithm for CPU scheduling,” Int. J. Comput. Sci. Eng., vol. 2, no. 7, pp. 2383–2385, 2010. [5] R. K. Yadav and A. Upadhayay, “A fresh loom for multilevel feedback queue scheduling algorithm,” Int. J. Adv. Eng. Sci., vol. 2, no. 3, pp. 21–23, 2012. [6] H. S. Behera, R. K. Naik, and S. Parida, “Improved multilevel feedback queue scheduling using dynamic time quantum and its performance analysis,” Int. J. Comput. Sci. Inf. Technol., vol. 3, pp. 3801–3807, 2012. [7] L. M. Ni and K. Hwang, “Optimal load balancing in a multiple processor system with many job classes,” IEEE Trans. Softw. Eng., no. 5, pp. 491–496, 1985. [8] D. M. Brooks et al., “Power-aware microarchitecture: Design and modeling challenges for next-generation microprocessors,” IEEE Micro, vol. 20, no. 6, pp. 26–44, 2000. [9] L. Wang, J. Tao, G. von Laszewski, and H. Marten, “Multicores in Cloud Computing: Research Challenges for Applications.,” JCP, vol. 5, no. 6, pp. 958–964, 2010. [10] C. L. Liu and J. W. Layland, “Scheduling algorithms for multiprogramming in a hard-real-time environment,” J. ACM, vol. 20, no. 1, pp. 46–61, 1973. [11] A. Burns, Preemptive priority based scheduling: An appropriate engineering approach. University of York, Department of Computer Science, 1993. [12] N. C. Audsley, A. Burns, R. I. Davis, K. W. Tindell, and A. J. Wellings, “Fixed priority pre-emptive scheduling: An historical perspective,” Real-Time Syst., vol. 8, no. 2–3, pp. 173–198, 1995. [13] S. B. N. Meghanathan, “A survey of contemporary real-time operating systems,” Informatica, vol. 29, no. 2, 2005. d R. Rajkumar, “Real-time operating systems,” Real-Time Syst., vol. 28, no. 2–3, pp. 237–253, 2004. 4] J. A. Stankovic and R. Rajkumar, “Real-time operating systems,” Real-Time Syst., vol. 28, no. 2–3, pp [15] M. Quigley et al., “ROS: an open-source Robot Operating System,” in ICRA workshop on open source software, 2009, vol. 3, no. 3.2, p. 5. [16] A. Silberschatz, P. B. Galvin, and G. Gagne, Operating system concepts essentials. John Wiley & Sons, Inc., 2014. Declaration of Competing Interest: The author declare no conflict of interest. Declaration of Competing Interest: The author declare no conflict of interest. 7. Conclusion Many functions are provided by an operating system like process management, memory management, file management, input/output management, networking, protection system and command interpreter system. An operating system is a very promising field for researchers because many issues need to be solved. Synchronization and scheduling contain most issues in OS; therefore, this paper focuses on the main algorithms in synchronization and scheduling with a focus on the issues and advantages in each algorithm. Journal of Studies in Science and Engineering. 2021, 1(2), 1-16. https://doi.org/10.53898/josse2021121 https://engiscience.com/index.php/josse M. Shakor 2021 16 There are many algorithms are available for CPU scheduling. But all algorithms have their deficiency and limitations. Each algorithm has some advantages or disadvantages. Furthermore, an overview of the new multiprocessor hardware and its effects on the operating system, scheduling and synchronization have been introduced. This paper, it is aimed to present a startup point for the researcher who wants to work on the synchronization and scheduling algorithms issues. There are many algorithms are available for CPU scheduling. But all algorithms have their deficiency and limitations. Each algorithm has some advantages or disadvantages. Furthermore, an overview of the new multiprocessor hardware and its effects on the operating system, scheduling and synchronization have been introduced. This paper, it is aimed to present a startup point for the researcher who wants to work on the synchronization and scheduling algorithms issues. References [17] E. W. Dijkstra, “Solution of a problem in concurrent programming control,” in Pioneers and Their Contributions to Software Engineering, Springer, 2001, pp. 289–294. [18] A. S. Tanenbaum, “Processes and Threads,” Mod. Oper. Syst., 2008. [18] A. S. Tanenbaum, “Processes and Threads,” Mod. Oper. Syst., 2008. Journal of Studies in Science and Engineering. 2021, 1(2), 1-16. https://doi.org/10.53898/josse2021121 https://engiscience.com/index.php/josse
https://openalex.org/W2113820196	https://bmcmedinformdecismak.biomedcentral.com/track/pdf/10.1186/s12911-015-0206-y	English	null	Prediction of delayed graft function after kidney transplantation: comparison between logistic regression and machine learning methods	BMC medical informatics and decision making	2,015	cc-by	7,610	* Correspondence: Alexander.Decruyenaere@UGent.be †Equal contributors 1Department of Nephrology, Ghent University Hospital, Ghent, Belgium Full list of author information is available at the end of the article © 2015 Decruyenaere et al. 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. Prediction of delayed graft function after kidney transplantation: comparison between logistic regression and machine learning methods Alexander Decruyenaere1*†, Philippe Decruyenaere1†, Patrick Peeters1, Frank Vermassen2, Tom Dhaene3 and Ivo Couckuyt3 Decruyenaere et al. BMC Medical Informatics and Decision Making (2015) 15:83 DOI 10.1186/s12911-015-0206-y Decruyenaere et al. BMC Medical Informatics and Decision Making (2015) 15:83 DOI 10.1186/s12911-015-0206-y RESEARCH ARTICLE Open Access Study cohort Kidney transplantation is the preferred treatment for patients with end-stage renal disease, improving survival, cardiovascular comorbidity and quality of life [1, 2]. Unfortunately, not every transplanted kidney is function- ing properly at the beginning. When ischemia/reperfu- sion injury is the cause of this early postoperative graft dysfunction, the term ‘delayed graft function’ (DGF) is used [3, 4]. DGF is diagnosed clinically after exclusion of other possible causes of early graft dysfunction, such as vascular thrombosis or hyperacute rejection [4, 5]. It is usually defined as the need for dialysis within the first week after transplantation [4]. The study cohort consists of consecutive adults (≥18 years) undergoing kidney transplantation from de- ceased donors at the Ghent University Hospital between January 1st, 2005 and December 31st, 2011. A total of 508 transplantations are performed. After exclusion of 11 transplantations, the study cohort consists of 497 transplantations. Reasons for exclusion are death of recipient or graft loss within the first week after trans- plantation. DGF is defined as the need for dialysis within the first week after transplantation. This study is con- ducted in accordance with the Declaration of Helsinki and is approved by the Ethics Committee of Ghent Uni- versity Hospital. Due to the retrospective nature of this study, the need for informed consent is waived. Despite advances in pretreatment of donors and recip- ients, as well as in diagnostic and therapeutic modalities, the incidence of DGF has not decreased, nor have its short-term and long-term effects [6]. The incidence is possibly increasing, which might partly be explained by using more expanded-criteria donors and donors after cardiac death, as well as by selecting more recipients who are possibly more prone to DGF. The incidence of DGF with deceased donors varies from 2 to 50 %, de- pending on country, transplant center and the definition used. The incidence of DGF with living donors is lower and varies from 4 to 10 % [7]. y Fifty-five parameters are retrospectively collected as potential risk factors for DGF. Parameters related to donor include age, sex, body mass index, cytomegalovirus serology, length of stay in intensive care unit, terminal serum creatinine, subtype, terminal urine output, terminal systolic and diastolic blood pressure, pretreatment with dopamine/dobutamine/epinephrine/norepinephrine, ter- minal central venous pressure, diabetes mellitus, history of hypertension, hypotensive episodes during pre-explanta- tion period, graft atherosclerosis (assessment by explant surgeon), and graft quality (assessment by explant sur- geon). Abstract BMC Medical Informatics and Decision Making (2015) 15:83 Decruyenaere et al. BMC Medical Informatics and Decision Making (2015) 15:83 Page 2 of 10 Page 2 of 10 Page 2 of 10 Study cohort Parameters related to preservation and operation include preservation method, preservation solution, cold ischemia time, warm ischemia time, perioperative diuresis, perioperative graft reperfusion, donor-recipient sex, and donor-recipient cytomegalovirus serology. Parameters re- lated to recipient include age, sex, ethnicity, body mass index, cytomegalovirus serology, modality and duration of dialysis, panel reactive antibodies at time of transplant- ation and peak panel reactive antibodies, number of previ- ous kidney transplantations, human leukocyte antigen mismatches, preoperative systolic and diastolic blood pressure at time of transplantation, diabetes mellitus, lipid levels (triglycerides, total cholesterol, high-density lipo- protein and low-density lipoprotein) at time of trans- plantation, pulmonary hypertension (systolic pulmonary artery pressure >35 mmHg during pretransplant evalu- ation period), iliac artery atheromatosis or stenosis (imaging studies during pretransplant evaluation period or assessment during transplantation), reduced cardiac function (ejection fraction <40 % during pretransplant evaluation period using echocardiography or coronary catheterization), impaired effective circulating volume (clinical assessment at time of transplantation), abdom- inal compartment syndrome (clinical assessment at time of transplantation), anti-thymocyte globulin induction therapy, acute calcineurin inhibitor toxicity (serum level above the recommended therapeutic range), urinary tract obstruction The short-term and long-term consequences of DGF are increasingly being documented. Firstly, DGF has an adverse impact on the immediate post-transplant course by causing prolonged hospitalization and rehabilitation, and higher transplantation costs [8, 9]. Secondly, it is associated with an increased rate of acute rejection and with reduced long-term graft function [10]. Finally, it leads to long-term graft loss [10], independent of the increased risk of acute rejection [11, 12], and reduced patient survival [13]. Because of the deleterious consequences, several pre- dictive models for DGF have been developed within the last few years. To date, four risk prediction models have been developed using logistic regression [14–17]. How- ever, machine learning methods are also effective to detect new risk factors and to achieve acceptable predictive ac- curacy [18, 19]. Brier et al. [20] and Santori et al. [21] have already demonstrated that neural networks have higher sensitivity but lower specificity than logistic regression in the prediction of DGF. Other studies suggest that neural networks [22] and tree-based models [23] also have higher sensitivity but lower specificity than Cox regression in the prediction of graft survival. Consistently, another tree- based model [24] and a Bayesian belief network [25] achieve reasonable predictive accuracy for graft survival. Abstract Background: Predictive models for delayed graft function (DGF) after kidney transplantation are usually developed using logistic regression. We want to evaluate the value of machine learning methods in the prediction of DGF. Methods: 497 kidney transplantations from deceased donors at the Ghent University Hospital between 2005 and 2011 are included. A feature elimination procedure is applied to determine the optimal number of features, resulting in 20 selected parameters (24 parameters after conversion to indicator parameters) out of 55 retrospectively collected parameters. Subsequently, 9 distinct types of predictive models are fitted using the reduced data set: logistic regression (LR), linear discriminant analysis (LDA), quadratic discriminant analysis (QDA), support vector machines (SVMs; using linear, radial basis function and polynomial kernels), decision tree (DT), random forest (RF), and stochastic gradient boosting (SGB). Performance of the models is assessed by computing sensitivity, positive predictive values and area under the receiver operating characteristic curve (AUROC) after 10-fold stratified cross-validation. AUROCs of the models are pairwise compared using Wilcoxon signed-rank test. Results: The observed incidence of DGF is 12.5 %. DT is not able to discriminate between recipients with and without DGF (AUROC of 52.5 %) and is inferior to the other methods. SGB, RF and polynomial SVM are mainly able to identify recipients without DGF (AUROC of 77.2, 73.9 and 79.8 %, respectively) and only outperform DT. LDA, QDA, radial SVM and LR also have the ability to identify recipients with DGF, resulting in higher discriminative capacity (AUROC of 82.2, 79.6, 83.3 and 81.7 %, respectively), which outperforms DT and RF. Linear SVM has the highest discriminative capacity (AUROC of 84.3 %), outperforming each method, except for radial SVM, polynomial SVM and LDA. However, it is the only method superior to LR. Conclusions: The discriminative capacities of LDA, linear SVM, radial SVM and LR are the only ones above 80 %. None of the pairwise AUROC comparisons between these models is statistically significant, except linear SVM outperforming LR. Additionally, the sensitivity of linear SVM to identify recipients with DGF is amongst the three highest of all models. Due to both reasons, the authors believe that linear SVM is most appropriate to predict DGF. Keywords: Decision trees, Delayed graft function, Discriminant analysis, Kidney transplantation, Logistic models, Machine learning, Predictive analysis, ROC curve, Sensitivity and specificity, Support vector machines Decruyenaere et al. BMC Medical Informatics and Decision Making (2015) 15:83 Decruyenaere et al. Study cohort In this study, the goal is therefore to analyze and dis- cuss the performance of different modeling techniques in the prediction of DGF and to identify which method is most suited to the task at hand. Page 3 of 10 Decruyenaere et al. BMC Medical Informatics and Decision Making (2015) 15:83 quadratic discriminant function, which contains second order terms. (assessment by surgeon during revision), and pretransplant transfusion. Categorical parameters with more than two pos- sible values are converted to indicator parameters (dummy variables) as required by most of the pre- dictive models. Support vector machines (SVMs) are sparse kernel machines, a type of models that rely only on a subset of the data (the support vectors) to predict unknown class labels. SVMs separate input data using a good-fitting hyperplane. Kernels can be used to transform this hyper- plane into a non-linear input separator. We chose a lin- ear, a radial basis function and a polynomial kernel. Feature selection Feature (or variable) selection is a process of determin- ing a subset of relevant parameters with respect to the predictive models. Many parameters might be irrelevant or contribute very little to the predictive models. Irrele- vant parameters can actually degrade the prediction. Hence, it is crucial to make a good selection of the most influential subset of parameters. A decision tree (DT) separates the data (the parent node) into two subsets (the child nodes) by the best splitting feature. The two resulting subsets become the new parent nodes, which are subsequently split further into two child nodes. This procedure continues until all observations are classified. In this study a recursive feature elimination procedure is used based on 10-fold stratified cross-validation [26]. The relative importance of the features is ranked using an external model, i.e., the coefficients of a logistic re- gression model. The full feature set is then iteratively pruned by removing the feature with the lowest import- ance until the 10-fold stratified cross-validation score decreases significantly, resulting in 24 selected parame- ters (two categorical parameters out of 20 selected parameters both have three possible values and are con- verted to three indicator parameters, resulting in a total of 24 selected parameters). Random forest (RF) is an ensemble machine learning method based on the construction of multiple decision trees. The main underlying technique is bootstrap aggre- gating (bagging). In each decision tree, a data point falls into a particular leaf depending on its features and is assigned a prediction. The predictions of the data points are then averaged. RF has a built-in feature selection system and allows for joint features, making it not only an additive model but also a multiplicative one. Stochastic gradient boosting (SGB) constructs additive regression tree models sequentially to fit pseudo-residuals of previous cumulative models. This stepwise manner combines the performance of weak learners (i.e., regres- sion trees here) iteratively into a strong learner with high accuracy. Statistical models h d d d The reduced data set of 24 parameters is fitted using 9 distinct types of predictive models: logistic regression, linear discriminant analysis, quadratic discriminant ana- lysis, support vector machines (using linear, radial basis function and polynomial kernels), decision tree, random forest and stochastic gradient boosting. An exhaustive grid search is used based on 10-fold stratified cross- validation to determine the optimal hyper-parameters of each predictive model. The hyper-parameters that are optimized are presented in Table 1 with the optimal values in bold. The hyper-parameters that are not de- scribed in this table are set to the default values used in the scikit-learn library [27]. As RF has a built-in feature selection system, the full data set of all collected parameters is also fitted using RF. By doing this, we can compare the performance between the RF fitted on the reduced data set and the RF fitted on the full data set, to evaluate if the recursive feature elimination procedure influences the built-in feature selection of RF. Model validation f f Performance of the models is assessed by computing the diagnostic test characteristics, including sensitivity and positive predictive value (PPV), and by evaluating the discriminative capacity, using the area under the receiver operating characteristic curve (AUROC), which mea- sures how well the relative ranking of the individual risk is in substantially the correct order (observed incidence in those with higher predicted risks are higher). Logistic regression (LR) is a linear model that assumes that the targets follow a Gaussian distribution. A predic- tion on a transplantation x is made using y(x) = wTx, where w is the weight vector being learned. Linear discriminant analysis (LDA) produces an opti- mally weighted linear function of chosen log-transformed markers and the discriminating threshold value minimizes the expected number of misclassifications under the nor- mal model. 10-fold stratified cross-validation is used to obtain a bet- ter generalization estimate of the performance. In 10-fold stratified cross-validation, the data set is partitioned into ten equal size folds such that each fold contains roughly the same proportion of ‘DGF’ and ‘no DGF’ class labels. Of the ten folds, a single fold is retained as the validation data for testing the model, and the remaining nine folds Quadratic discriminant analysis (QDA) is related to LDA. Unlike LDA however, there is no assumption that the covariance of each class is identical. This produces a Decruyenaere et al. Model validation f f BMC Medical Informatics and Decision Making (2015) 15:83 Page 4 of 10 Page 4 of 10 Table 1 Optimal hyper-parameters after exhaustive grid search Statistical method Hyper-parameter Values Decision tree Class weights auto, 0 to 0.20 and 1 to 0.80, 0 to 0.10 and 1 to 0.90, 0 to 0.05 and 1 to 0.95 Maximum depth 1 to 10 (8) Minimum samples split 2 to nVars+1 (18) Maximum features auto, sqrt, log2 Random forest Number of estimators 1000 Class weights auto, 0 to 0.20 and 1 to 0.80, 0 to 0.10 and 1 to 0.90, 0 to 0.05 and 1 to 0.95 Maximum depth 1 to 10 (9) Minimum samples split 2 to nVars+1 (24) Maximum features auto, sqrt, log2 Random forest (full) Number of estimators 1000 Class weights auto, 0 to 0.20 and 1 to 0.80, 0 to 0.10 and 1 to 0.90, 0 to 0.05 and 1 to 0.95 Maximum depth 1 to 10 (1) Minimum samples split 2 to nVars+1 (63) Maximum features auto, sqrt, log2 Gradient boosting Number of estimators 1000 Maximum depth 1 to 10 (1) Minimum samples split 2 to nVars+1 (9) Maximum features auto, sqrt, log2 Learning rate 0.1, 0.05, 0.02, 0.01 LDA Number of components None or 1 to nVars +1 QDA Regularizing parameter 0 to 1 (0.89) Linear SVM Class weights auto, 0 to 0.20 and 1 to 0.80, 0 to 0.10 and 1 to 0.90, 0 to 0.05 and 1 to 0.95 C 0.001, 0.01, 0.1, 1, 10, 100, 1000 Radial SVM Class weights auto, 0 to 0.20 and 1 to 0.80, 0 to 0.10 and 1 to 0.90, 0 to 0.05 and 1 to 0.95 C 0.001, 0.01, 0.1,1, 10, 100, 1000 Gamma 0.1, 0.01, 0.001, 0.0001 Polynomial SVM Class weights auto, 0 to 0.20 and 1 to 0.80, 0 to 0.10 and 1 to 0.90, 0 to 0.05 and 1 to 0.95 C 0.001, 0.01, 0.1,1, 10, 100, 1000 Gamma 0.1, 0.01, 0.001, 0.0001 Logistic regression Class weights auto, 0 to 0.20 and 1 to 0.80, 0 to 0.10 and 1 to 0.90, 0 to 0.05 and 1 to 0.95 C 0.001, 0.01, 0.1,1, 10, 100, 1000 The hyper-parameters that are not described in this table are set to the default values used in the scikit-learn library [27] Abbreviations: LDA linear discriminant analysis, QDA quadratic discriminant analysis, SVM support vector machine auto, 0 to 0.20 and 1 to 0.80, 0 to 0.10 and 1 to 0.90, 0 to 0.05 and 1 to 0.95 C 0.001, 0.01, 0.1,1, 10, 100, 1000 0.001, 0.01, 0.1,1, 10, 100, 1000 0.001, 0.01, 0.1,1, 10, 100, 1000 The hyper-parameters that are not described in this table are set to the default values used in the scikit-learn library [27 Abbreviations: LDA linear discriminant analysis, QDA quadratic discriminant analysis, SVM support vector machine Descriptive statistics The most relevant donor, preservation/operation, and recipient characteristics are presented in Table 2. After exclusion, 497 transplantations are used for the analysis, consisting of 432 unique donors (362 donated a single kidney to a recipient of our center and the other kidney to a recipient of a different center, 5 donated both kid- neys to the same recipient of our center, and 65 donated both kidneys to different recipients of our center) and 496 unique recipients (1 recipient underwent two kidney transplantations at different times from deceased donors during the study period). The observed incidence of DGF is 12.5 % (62/497). This imbalance in the data set is addressed by assigning more weight to the ‘DGF’ class during the learning phase of the predictive models. Only 11 (categorical) parameters out of the 55 retrospectively collected parameters are in- complete and contain missing values for a number of transplantations. The most frequent occurring value, which is the ‘normal’ category, is used to fill in these miss- ing values. This is a safe assumption, because ‘abnormal’ values for risk factors are more likely to be emphasized and registered in the electronic medical records. However, ‘normal’ values are not always routinely registered in the electronic medical records and are retrospectively consid- ered as missing values. Model comparison are used as training data. The cross-validation process is then repeated ten times, with each of the ten folds used exactly once as the validation data. The ten results from the folds are averaged to produce a single estimation. The advantage is that all observations are used for both training and validation, and each observation is used for validation exactly once. Subsequently, the models are pairwise compared. For each model, the AUROC is computed in each of the ten folds. The ten values for the AUROC of one model are compared with the values of another model using the two-sided Wilcoxon signed-rank test at 5 % significance level. Page 5 of 10 Page 5 of 10 Decruyenaere et al. BMC Medical Informatics and Decision Making (2015) 15:83 Table 2 Baseline characteristics (n = 497) All computations are carried out using Python, specific- ally in the SciPy environment using the scikit-learn library [27]. Continuous data are presented as mean ± standard deviation and categorical data are reported as percentages. Counts are put in parentheses. and only slightly lower sensitivity in identifying recipients without DGF (94.7 and 89.9 %, respectively), resulting in higher discriminative capacity (AUROC of 82 2 and Table 2 Baseline characteristics (n = 497) Donor Sex male 60.4 % (300) female 39.6 % (197) Subtype DBD 90.3 % (449) DCD 9.7 % (48) Age (year) 42.6 ± 14.77 Terminal SCr (mg/dL) 0.878 ± 0.4757 Preservation/Operation Preservation solution HTK 31.0 % (154) HTK + UW 0.2 % (1) UW 68.6 % (341) missing 0.2 % (1) CIT (hour) 14.19 ± 4.328 WIT (min) 22.3 ± 7.09 Recipient Sex male 66.6 % (331) female 33.4 % (166) Modality of dialysis hemodialysis 71.2 % (354) peritoneal dialysis 22.7 % (113) pre-emptive 6.0 % (30) HLA mismatches 0 8.9 % (44) 1 7.8 % (39) 2 26.4 % (131) 3 40.8 % (203) 4 10.9 % (54) 5 4.0 % (20) 6 1.2 % (6) Age (year) 52.8 ± 11.68 Duration of dialysis (year) 2.7 ± 1.68 PRA at time of Tx (%) 2.7 ± 11.44 Abbreviations: CIT cold ischemia time, DBD donor after brain death, DCD donor after cardiac/circulatory death, HLA human leukocyte antigen, HTK histidine- tryptophan-ketoglutarate, PRA panel reactive antibody, SCr serum creatinine, Tx transplantation, UW University of Wisconsin, WIT warm ischemia time Model performance and comparison Diagnostic test characteristics and AUROCs after 10-fold stratified cross-validation are presented in Table 3. The re- ceiver operating characteristic curves and the p-values of the pairwise AUROC comparisons are presented in Figs. 1 and 2, respectively. The selected features and their respect- ive odds ratios (LR), Z-scores (linear SVM), and Gini index (RF fitted on the full data set) are presented in Table 4. DT is not able to discriminate between recipients with and without DGF (AUROC of 52.5 %) and is inferior to the other methods. Abbreviations: CIT cold ischemia time, DBD donor after brain death, DCD donor after cardiac/circulatory death, HLA human leukocyte antigen, HTK histidine- tryptophan-ketoglutarate, PRA panel reactive antibody, SCr serum creatinine, Tx transplantation, UW University of Wisconsin, WIT warm ischemia time As SGB and RF mainly have high sensitivity (98.8 and 96.3 %, respectively) and high PPVs (89.2 and 89.0 %, respectively) in identifying recipients without DGF, their discriminative capacity (AUROC of 77.2 and 73.9 %, re- spectively) is superior to DT. However, RF is still outper- formed by LDA, QDA, linear SVM, radial SVM and LR. SGB is only outperformed by linear SVM. and only slightly lower sensitivity in identifying recipients without DGF (94.7 and 89.9 %, respectively), resulting in higher discriminative capacity (AUROC of 82.2 and 79.6 %, respectively). Both LDA and QDA outperform DT and RF, but only QDA is inferior to linear SVM. Amongst all methods used, linear SVM, radial SVM and LR have the highest sensitivity in identifying recipients with LDA and QDA already have higher sensitivity in identi- fying recipients with DGF (27.6 and 37.6 %, respectively) Decruyenaere et al. Model performance and comparison BMC Medical Informatics and Decision Making (2015) 15:83 Page 6 of 10 Page 6 of 10 Table 3 Performance of the statistical methods after 10-fold stratified cross-validation Statistical method Sensitivity (%) PPV (%) AUROC (%) No DGF DGF No DGF DGF Decision tree 75.4 ± 6.64 29.5 ± 16.29 88.2 ± 2.73 14.2 ± 8.13 52.5 ± 8.55 Gradient boosting 98.8 ± 1.55 16.2 ± 12.94 89.2 ± 1.67 58.3 ± 38.19 77.2 ± 9.64 Random forest 96.3 ± 4.05 16.4 ± 14.92 89.0 ± 2.09 43.9 ± 38.19 73.9 ± 9.94 Random forest (full) 100.0 ± 0.00 0.0 ± 0.00 87.5 ± 0.64 0.0 ± 0.00 71.6 ± 12.38 LDA 94.7 ± 2.92 27.6 ± 15.10 90.2 ± 2.00 42.3 ± 19.94 82.2 ± 6.14 QDA 89.9 ± 5.35 37.6 ± 17.26 91.0 ± 2.55 37.9 ± 20.82 79.6 ± 7.55 Linear SVM 72.0 ± 6.29 83.8 ± 7.51 96.9 ± 1.34 30.6 ± 5.60 84.3 ± 4.11 Radial SVM 57.9 ± 7.45 88.8 ± 7.38 97.2 ± 1.87 23.6 ± 4.14 83.3 ± 4.05 Polynomial SVM 97.5 ± 1.90 10.9 ± 12.20 88.5 ± 1.14 24.0 ± 24.17 79.8 ± 5.33 Logistic regression 65.0 ± 8.25 85.5 ± 8.94 96.9 ± 1.84 26.5 ± 4.75 81.7 ± 5.82 Abbreviations: AUROC area under the receiver operating characteristic curve, DGF delayed graft function, LDA linear discriminant analysis, PPV positive predictive value, QDA quadratic discriminant analysis, SVM support vector machine Table 3 Performance of the statistical methods after 10-fold stratified cross-validation Abbreviations: AUROC area under the receiver operating characteristic curve, DGF delayed graft function, LDA linear discriminant analy value, QDA quadratic discriminant analysis, SVM support vector machine a under the receiver operating characteristic curve, DGF delayed graft function, LDA linear discriminant analysis, PPV positive predictive riminant analysis, SVM support vector machine DGF (83.8, 88.8 and 85.5 %, respectively), at the expense of identifying recipients without DGF (72.0, 57.9 and 65.0 %, respectively). However, their capability to identify both out- comes is reflected in a strong discriminative capacity (AUROC of 84.3, 83.3 and 81.7 %, respectively). Linear SVM outperforms each method, except for radial SVM, polynomial SVM and LDA. Radial SVM and LR outper- form DT and RF, but only LR is inferior to linear SVM. DGF (83.8, 88.8 and 85.5 %, respectively), at the expense of identifying recipients without DGF (72.0, 57.9 and 65.0 %, respectively). Model performance and comparison However, their capability to identify both out- comes is reflected in a strong discriminative capacity (AUROC of 84.3, 83.3 and 81.7 %, respectively). Linear SVM outperforms each method, except for radial SVM, polynomial SVM and LDA. Radial SVM and LR outper- form DT and RF, but only LR is inferior to linear SVM. The performance of polynomial SVM is similar to that of SGB and RF, with high sensitivity (97.5 %) and high PPV (88.5 %) in identifying recipients without DGF, resulting in an AUROC of 79.8 %. Polynomial SVM also outperforms DT. Unlike SGB and RF however, it is not inferior to any of the methods used. RF fitted on the full data set has a sensitivity of 100 % and a PPV of 87.5 % in identifying recipients without Fig. 1 Receiver operating characteristic curves after 10-fold stratified cross-validation. Abbreviations: AUROC area under the receiver operating Fig. 1 Receiver operating characteristic curves after 10-fold stratified cross-validation. Abbreviations: AUROC area under the receiver operating characteristic curve, LDA linear discriminant analysis, QDA quadratic discriminant analysis, SVM support vector machine Fig. 1 Receiver operating characteristic curves after 10-fold stratified cross-validation. Abbreviations: AUROC area under the receiver operating characteristic curve, LDA linear discriminant analysis, QDA quadratic discriminant analysis, SVM support vector machine Decruyenaere et al. BMC Medical Informatics and Decision Making (2015) 15:83 Page 7 of 10 Page 7 of 10 Fig. 2 P-values (%) of pairwise model comparison using Wilcoxon signed-rank test. Abbreviations: LDA linear discriminant analysis, QDA quadratic discriminant analysis, SVM support vector machine Fig. 2 P-values (%) of pairwise model comparison using Wilcoxon signed-rank test. Abbreviations: LDA linear discriminant analysis, QDA quadratic discriminant analysis, SVM support vector machine Fig. 2 P-values (%) of pairwise model comparison using Wilcoxon signed-rank test. Abbreviations: LDA linear discriminant analysis, QDA quadratic discriminant analysis, SVM support vector machine Fig. 2 P-values (%) of pairwise model comparison using Wilcoxon signed-rank test. Abbreviations: LDA linear discriminant analysis, QDA quadratic discriminant analysis, SVM support vector machine SVM, polynomial SVM and LDA. However, it is the only method superior to LR. DGF, resulting in an AUROC of 71.6 %. It is superior to DT, which is fitted on the reduced data set and has no discriminative capacity, and non-inferior to RF fitted on the reduced data set. However, RF fitted on the full data set is inferior to each of the other methods used. Model performance and comparison The AUROC focuses solely on the predictive accuracy of a model. As such, it cannot tell us whether the model is worth using in clinical practice, because it does not in- corporate information on consequences. The method with maximal accuracy is not necessarily the best to choose. This choice should depend on the disadvantages or costs of not identifying a recipient with DGF as op- posed to incorrectly predicting DGF in a recipient who will not develop it [28]. The advantages of an early hypo- thetic treatment should be weighed against possible iat- rogenic damage and unnecessary additional costs. If we assume that the damage of an unnecessary treatment of DGF (a false-positive result) is limited, a more sensitive method should be used. If an unnecessary treatment is harmful, a more specific method should be used. Of course the trade-off between sensitivity and specificity should be kept in mind: a very sensitive method is use- less when it is not specific enough and vice versa [29]. Discussion The risk prediction of DGF may be important in pre- venting its deleterious short-term and long-term conse- quences. To date, four predictive models are developed as a clinical tool to quantify the risk for DGF [14–17]. All models are developed using LR. We compared in this study several machine learning methods, including LR, in terms of their predictive accuracy for DGF. There are no studies that have used DT, SGB, RF, LDA, QDA or SVM in the prediction of DGF. In our study, DT is not able to discriminate between recipients with and without DGF, and is inferior to the other methods. SGB, RF and polynomial SVM are mainly able to identify recipients without DGF and only outperform DT. Despite lower sensitivity in varying degrees to identify recipients without DGF, LDA, QDA, radial SVM and LR also have the ability to identify recipients with DGF, resulting in higher discriminative capacity, which outperforms DT and RF. Linear SVM has the highest discriminative capacity (AUROC of 84.3 %), outperforming each method, except for radial Currently, the management of DGF consists of a careful follow-up. Besides sonographic evaluation and precise bio- chemical monitoring, a biopsy is often performed, which is costly and invasive, possibly damaging the graft. Because of the complex and multifactorial characteristics of DGF, a standard therapy or drug does not yet exist [30]. Although a biopsy might be harmful, this is outweighed by the Decruyenaere et al. BMC Medical Informatics and Decision Making (2015) 15:83 Page 8 of 10 Page 8 of 10 Table 4 Weights of the selected features Feature Odds ratio (LR)a Z-score (linear SVM)a Gini index (RF)b Donor Age (per 1 year) 1.060 0.744 0.037 (#9) BMI (per 1 kg/m2) 0.751 −1.700 0.023 (#20) Terminal SCr (per 1 mg/dL) 6.512 1.126 0.024 (#17.5) Hypotensive episodes: yes vs. no 1.784 0.165 0.001 (#48.5) Diabetes mellitus: yes vs. no 0.013 −1.041 0.001 (#48.5) History of hypertension: yes vs. no 3.585 0.940 0.011 (#28) Donor after cardiac death: yes vs. no 25.789 1.534 0.080 (#1) Preservation/Operation Machine perfusion: yes vs. no 0.003 −1.078 0.000 (#60) Perioperative graft reperfusionc 0.740 −0.844 0.027 (#14.5) Preservation solution HTK + UW 0.00005 −0.510 0.000 (#60) UW 0.080 −1.557 0.016 (#25) HTK 0.050 −1.725 0.007 (#32.5) Male donor-to-female recipient: yes vs. Discussion no 0.352 −0.750 0.019 (#23) Recipient BMI (per 1 kg/m2) 1.144 0.941 0.054 (#4) Duration of dialysis (per 1 day) 1.0005 0.324 0.057 (#3) PRA at time of Tx (per 1 %) 0.977 −0.557 0.008 (#30.5) Peak PRA (per 1 %) 1.017 0.585 0.025 (#16) Acute CNI toxicity: yes vs. no 22.044 0.964 0.007 (#32.5) Reduced cardiac function: yes vs. no 5.570 0.897 0.033 (#13) Impaired ECV: yes vs. no 0.003 −1.141 0.000 (#60) Urinary tract obstruction: yes vs. no 6.638 0.942 0.004 (#38.5) Iliac artery normal 1.520 0.221 0.001 (#48.5) atheromatosis 2.389 0.573 0.006 (#34.5) stenosis 28.465 0.948 0.037 (#9) aFitted on the reduced data set bFitted on the full data set. Tied rank amongst all 68 features is given in parentheses cPerioperative graft reperfusion is an ordinal feature (poor – patchy – moderate – good) Abbreviations: BMI body mass index, CNI calcineurin inhibitor toxicity, ECV effective circulating volume, HTK histidine-tryptophan-ketoglutarate, LR logistic regression, PRA panel reactive antibody, RF random forest, SCr serum creatinine, SVM support vector machine, Tx transplantation, UW University of Wisconsin bFitted on the full data set. Tied rank amongst all 68 features is given in parentheses cPerioperative graft reperfusion is an ordinal feature (poor – patchy – moderate – good) Abbreviations: BMI body mass index, CNI calcineurin inhibitor toxicity, ECV effective circulating volume, HTK histidine-tryptophan-ketoglutarate, LR logistic regression, PRA panel reactive antibody, RF random forest, SCr serum creatinine, SVM support vector machine, Tx transplantation, UW University of Wisconsin prediction of DGF. The sensitivity of linear SVM to iden- tify recipients with DGF (83.8 %) is amongst the three highest of all methods used. Only radial SVM and LR have a slightly higher sensitivity (88.8 and 85.5 %, respectively). Due to both reasons, the authors believe that linear SVM is most appropriate to predict DGF. potential benefit of an early management, because DGF has deleterious short-term and long-term consequences. To date, a more sensitive method is therefore preferred. In our study, linear SVM, radial SVM and LR have the high- est sensitivity in identifying recipients with DGF (83.8, 88.8 and 85.5 %, respectively). 72.0 % of the recipients who will not develop DGF are identified. These recipients can undergo the kidney trans- plantation without the need for a more precise monitor- ing. Only 3.1 % will still develop DGF. 83.8 % of the recipients who will develop DGF are identified. Conclusions Ni di i Nine distinct types of predictive models for DGF are considered. The discriminative capacities of LDA, linear SVM, radial SVM and LR are the only ones above 80 %. None of the pairwise AUROC comparisons between these models is statistically significant, except linear SVM outperforming LR. Additionally, a method with higher sensitivity is preferred over a method with higher specificity in the prediction of DGF, because the damage of an unnecessary biopsy is outweighed by the potential benefit of an early management. The sensitivity of linear SVM to identify recipients with DGF is amongst the three highest of all models. Due to both reasons, the authors believe that linear SVM is most appropriate to predict DGF. 6. Siedlecki A, Irish W, Brennan DC. Delayed graft function in the kidney transplant. Am J Transplant. 2011;11(11):2279–96. 7. Perico N, Cattaneo D, Sayegh MH, Remuzzi G. Delayed graft function in kidney transplantation. Lancet. 2004;364(9447):1814–27. y p 8. Matas AJ, Gillingham KJ, Elick BA, Dunn DL, Gruessner RW, Payne WD, et al. Risk factors for prolonged hospitalization after kidney transplants. Clin Transpl. 1997;11(4):259–64. 9. Rosenthal JT, Danovitch GM, Wilkinson A, Ettenger RB. The high cost of delayed graft function in cadaveric renal transplantation. Transplantation. 1991;51(5):1115–8. 9. Rosenthal JT, Danovitch GM, Wilkinson A, Ettenger RB. The high cost of delayed graft function in cadaveric renal transplantation. Transplantation. 1991;51(5):1115–8. 10. Yarlagadda SG, Coca SG, Formica Jr RN, Poggio ED, Parikh CR. Association between delayed graft function and allograft and patient survival: a systematic review and meta-analysis. Nephrol Dial Transplant. 2009;24(3):1039–47. 10. Yarlagadda SG, Coca SG, Formica Jr RN, Poggio ED, Parikh CR. Association between delayed graft function and allograft and patient survival: a systematic review and meta-analysis. Nephrol Dial Transplant. 2009;24(3):1039–47. 11. Shoskes DA, Cecka JM. Deleterious effects of delayed graft function in cadaveric renal transplant recipients independent of acute rejection. Transplantation. 1998;66(12):1697–701. 11. Shoskes DA, Cecka JM. Deleterious effects of delayed graft function in cadaveric renal transplant recipients independent of acute rejection. Transplantation. 1998;66(12):1697–701. 12. Troppmann C, Gruessner AC, Gillingham KJ, Sutherland DE, Matas AJ, Gruessner RW. Impact of delayed function on long-term graft survival after solid organ transplantation. Transplant Proc. 1999;31(1–2):1290–2. 12. Troppmann C, Gruessner AC, Gillingham KJ, Sutherland DE, Matas AJ, Gruessner RW. Impact of delayed function on long-term graft survival after solid organ transplantation. Transplant Proc. 1999;31(1–2):1290–2. Received: 16 April 2015 Accepted: 30 September 2015 Received: 16 April 2015 Accepted: 30 September 2015 Received: 16 April 2015 Accepted: 30 September 2015 Authors’ information 2010;42(4):1134–6. 19. Lasserre J, Arnold S, Vingron M, Reinke P, Hinrichs C. Predicting the outcome of renal transplantation. J Am Med Inform Assoc. 2012;19(2):255–62. 20. Brier ME, Ray PC, Klein JB. Prediction of delayed renal allograft function using an artificial neural network. Nephrol Dial Transplant. 2003;18(12):2655–9. 21. Santori G, Fontana I, Valente U. Application of an artificial neural network model to predict delayed decrease of serum creatinine in pediatric patients after kidney transplantation. Transplant Proc. 2007;39(6):1813–9. Alexander Decruyenaere and Philippe Decruyenaere are the joint first authors. 19. Lasserre J, Arnold S, Vingron M, Reinke P, Hinrichs C. Predicting the outcome of renal transplantation. J Am Med Inform Assoc. 2012;19(2):255–62. 19. Lasserre J, Arnold S, Vingron M, Reinke P, Hinrichs C. Predicting the outcome of renal transplantation. J Am Med Inform Assoc. 2012;19(2):255–62. 20. Brier ME, Ray PC, Klein JB. Prediction of delayed renal allograft function using an artificial neural network. Nephrol Dial Transplant. 2003;18(12):2655–9. 20. Brier ME, Ray PC, Klein JB. Prediction of delayed renal allograft function using an artificial neural network. Nephrol Dial Transplant. 2003;18(12):2655–9. References ll 1. Purnell TS, Auguste P, Crews DC, Lamprea-Montealegre J, Olufade T, Greer R, et al. Comparison of life participation activities among adults treated by hemodialysis, peritoneal dialysis, and kidney transplantation: a systematic review. Am J Kidney Dis. 2013;62(5):953–73. 1. Purnell TS, Auguste P, Crews DC, Lamprea-Montealegre J, Olufade T, Greer R, et al. Comparison of life participation activities among adults treated by hemodialysis, peritoneal dialysis, and kidney transplantation: a systematic review. Am J Kidney Dis. 2013;62(5):953–73. 2. Tonelli M, Wiebe N, Knoll G, Bello A, Browne S, Jadhav D, et al. Systematic review: kidney transplantation compared with dialysis in clinically relevant outcomes. Am J Transplant. 2011;11(10):2093–109. p 3. Cavaille-Coll M, Bala S, Velidedeoglu E, Hernandez A, Archdeacon P, Gonzalez G, et al. Summary of FDA workshop on ischemia reperfusion inju in kidney transplantation. Am J Transplant. 2013;13(5):1134–48. y p p 4. Yarlagadda SG, Coca SG, Garg AX, Doshi M, Poggio E, Marcus RJ, et al. Marked variation in the definition and diagnosis of delayed graft function: systematic review. Nephrol Dial Transplant. 2008;23(9):2995–3003. y p p 5. Sharif A, Borrows R. Delayed graft function after kidney transplantation: the clinical perspective. Am J Kidney Dis. 2013;62(1):150–8. 6. Siedlecki A, Irish W, Brennan DC. Delayed graft function in the kidney transplant. Am J Transplant. 2011;11(11):2279–96. 7. Perico N, Cattaneo D, Sayegh MH, Remuzzi G. Delayed graft function in kidney transplantation. Lancet. 2004;364(9447):1814–27. 8 Matas AJ Gillingham KJ Elick BA Dunn DL Gruessner RW Payne WD et al 5. Sharif A, Borrows R. Delayed graft function after kidney transplantation: the clinical perspective. Am J Kidney Dis. 2013;62(1):150–8. Authors’ contributions d d d d AD and PD designed study, collected data, interpreted statistical analysis and drafted paper. Both authors contributed equally to this study and are the joint first authors. PP, FV and TD designed study and revised paper. IC designed study, performed and interpreted statistical analysis, and revised paper. All authors read and approved the final manuscript. 17. Zaza G, Ferraro PM, Tessari G, Sandrini S, Scolari MP, Capelli I, et al. Predictive model for delayed graft function based on easily available pre-renal transplant variables. Intern Emerg Med. 2015;10(2):135–41. 18. Greco R, Papalia T, Lofaro D, Maestripieri S, Mancuso D, Bonofiglio R. Decisional trees in renal transplant follow-up. Transplant Proc. 2010;42(4):1134–6. Competing interests p g The authors declare that they have no competing interests. 16. Chapal M, Le Borgne F, Legendre C, Kreis H, Mourad G, Garrigue V, et al. A useful scoring system for the prediction and management of delayed graft function following kidney transplantation from cadaveric donors. Kidney Int. 2014;86(6):1130–9. 16. Chapal M, Le Borgne F, Legendre C, Kreis H, Mourad G, Garrigue V, et al. A useful scoring system for the prediction and management of delayed graft function following kidney transplantation from cadaveric donors. Kidney Int. 2014;86(6):1130–9. Abbreviations 13. Tapiawala SN, Tinckam KJ, Cardella CJ, Schiff J, Cattran DC, Cole EH, et al. Delayed graft function and the risk for death with a functioning graft. J Am Soc Nephrol. 2010;21(1):153–61. 13. Tapiawala SN, Tinckam KJ, Cardella CJ, Schiff J, Cattran DC, Cole EH, et al. Delayed graft function and the risk for death with a functioning graft. J Am Soc Nephrol. 2010;21(1):153–61. AUROC: Area under the receiver operating characteristic curve; DGF: Delayed graft function; DT: Decision tree; LDA: Linear discriminant analysis; LR: Logistic regression; PPV: Positive predictive value; QDA: Quadratic discriminant analysis; RF: Random forest; SGB: Stochastic gradient boosting; SVM: Support vector machine. 14. Jeldres C, Cardinal H, Duclos A, Shariat SF, Suardi N, Capitanio U, et al. Prediction of delayed graft function after renal transplantation. Can Urol Assoc J. 2009;3(5):377–82. 14. Jeldres C, Cardinal H, Duclos A, Shariat SF, Suardi N, Capitanio U, et al. Prediction of delayed graft function after renal transplantation. Can Urol Assoc J. 2009;3(5):377–82. 15. Irish WD, Ilsley JN, Schnitzler MA, Feng S, Brennan DC. A risk prediction model for delayed graft function in the current era of deceased donor renal transplantation. Am J Transplant. 2010;10(10):2279–86. 15. Irish WD, Ilsley JN, Schnitzler MA, Feng S, Brennan DC. A risk prediction model for delayed graft function in the current era of deceased donor renal transplantation. Am J Transplant. 2010;10(10):2279–86. Discussion These re- cipients will have to be precisely monitored after kidney transplantation, making an early identification of graft To sum up, the discriminative capacities of LDA, linear SVM, radial SVM and LR are the only ones above 80 % (82.2, 84.3, 83.3 and 81.7 %, respectively). None of the pairwise AUROC comparisons between these models is statistically significant, except linear SVM outperforming LR. Additionally, a method with higher sensitivity is preferred over a method with higher specificity in the Decruyenaere et al. BMC Medical Informatics and Decision Making (2015) 15:83 Page 9 of 10 Page 9 of 10 dysfunction possible. 69.4 % of all positively identified re- cipients will eventually not develop DGF. AUROC: Area under the receiver operating characteristic curve; DGF: Delayed graft function; DT: Decision tree; LDA: Linear discriminant analysis; LR: Logistic regression; PPV: Positive predictive value; QDA: Quadratic discriminant analysis; RF: Random forest; SGB: Stochastic gradient boosting; SVM: Support vector machine. Author details 1 1Department of Nephrology, Ghent University Hospital, Ghent, Belgium. 2Department of Thoracic and Vascular Surgery, Ghent University Hospital, Ghent, Belgium. 3Department of Information Technology (INTEC), Ghent University - iMinds, Ghent, Belgium. Our study does have limitations. Firstly, our sample size of approximately 500 transplantations is lower than in the existing models. It is known that machine learning tech- niques generally benefit from a large amount of data, in- creasing their performance [19]. However, we benefited from the detailed and high-quality peritransplant data that could be collected, which is largely unavailable in regis- tries. Secondly, the incidence of DGF in our cohort is lower than in the existing models. This imbalance is ad- dressed by assigning more weight to the ‘DGF’ class dur- ing the learning phase of the predictive models. Thirdly, single-center models limit generalizability. However, we used cross-validation to attenuate the generalization error. Finally, our analysis included most, but not all, of the identified risk factors for DGF. Acknowledgements The authors would like to thank the transplant coordinators, with special thanks to Marc Van Der Vennet, for providing part of the data. The authors declare that they have not received any funding. 21. Santori G, Fontana I, Valente U. Application of an artificial neural network model to predict delayed decrease of serum creatinine in pediatric patients after kidney transplantation. Transplant Proc. 2007;39(6):1813–9. 21. Santori G, Fontana I, Valente U. Application of an artificial neural network model to predict delayed decrease of serum creatinine in pediatric patients after kidney transplantation. Transplant Proc. 2007;39(6):1813–9. Page 10 of 10 Page 10 of 10 Decruyenaere et al. BMC Medical Informatics and Decision Making (2015) 15:83 Decruyenaere et al. BMC Medical Informatics and Decision Making (2015) 15:83 22. Akl A, Ismail AM, Ghoneim M. Prediction of graft survival of living-donor kidney transplantation: nomograms or artificial neural networks? Transplantation. 2008;86(10):1401–6. 23. Goldfarb-Rumyantzev AS, Scandling JD, Pappas L, Smout RJ, Horn S. Prediction of 3-yr cadaveric graft survival based on pre-transplant variables in a large national dataset. Clin Transpl. 2003;17(6):485–97. 24. Krikov S, Khan A, Baird BC, Barenbaum LL, Leviatov A, Koford JK, et al. Predicting kidney transplant survival using tree-based modeling. ASAIO J. 2007;53(5):592–600. 25. Brown TS, Elster EA, Stevens K, Graybill JC, Gillern S, Phinney S, et al. Bayesian modeling of pretransplant variables accurately predicts kidney graft survival. Am J Nephrol. 2012;36(6):561–9. 26. Guyon I, Weston J, Barnhill S, Vapnik V. Gene selection for cancer classification using support vector machines. Mach Learn. 2002;46(1–3):389–422. 27. Pedregosa F, Varoquaux G, Gramfort A, Michel V, Thirion B, Grisel O, et al. Scikit-learn: machine learning in python. J Mach Learn Res. 2011;12:2825–30. 28. Vickers AJ, Elkin EB. Decision curve analysis: a novel method for evaluating prediction models. Med Decis Making. 2006;26(6):565–74. 29. Zweig MH, Campbell G. Receiver-operating characteristic (ROC) plots: a fundamental evaluation tool in clinical medicine. Clin Chem. 1993;39(4):561–77. 30. Powell JT, Tsapepas DS, Martin ST, Hardy MA, Ratner LE. Managing renal transplant ischemia reperfusion injury: novel therapies in the pipeline. Clin Transpl. 2013;27(4):484–91. 22. Akl A, Ismail AM, Ghoneim M. Prediction of graft survival of living-donor kidney transplantation: nomograms or artificial neural networks? Transplantation. 2008;86(10):1401–6. 23. Goldfarb-Rumyantzev AS, Scandling JD, Pappas L, Smout RJ, Horn S. Prediction of 3-yr cadaveric graft survival based on pre-transplant variables in a large national dataset. Clin Transpl. 2003;17(6):485–97. 24. Acknowledgements Krikov S, Khan A, Baird BC, Barenbaum LL, Leviatov A, Koford JK, et al. Predicting kidney transplant survival using tree-based modeling. ASAIO J. 2007;53(5):592–600. 25. Brown TS, Elster EA, Stevens K, Graybill JC, Gillern S, Phinney S, et al. Bayesian modeling of pretransplant variables accurately predicts kidney graft survival. Am J Nephrol. 2012;36(6):561–9. 26. Guyon I, Weston J, Barnhill S, Vapnik V. Gene selection for cancer classification using support vector machines. Mach Learn. 2002;46(1–3):389–422. 27. Pedregosa F, Varoquaux G, Gramfort A, Michel V, Thirion B, Grisel O, et al. Scikit-learn: machine learning in python. J Mach Learn Res. 2011;12:2825–30. 28. Vickers AJ, Elkin EB. Decision curve analysis: a novel method for evaluating prediction models. Med Decis Making. 2006;26(6):565–74. 29. Zweig MH, Campbell G. Receiver-operating characteristic (ROC) plots: a fundamental evaluation tool in clinical medicine. Clin Chem. 1993;39(4):561–77. 30. Powell JT, Tsapepas DS, Martin ST, Hardy MA, Ratner LE. Managing renal transplant ischemia reperfusion injury: novel therapies in the pipeline. Clin Transpl. 2013;27(4):484–91. 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https://openalex.org/W2765965050	https://jecs.pl/index.php/jecs/article/download/10.15503.jecs20172.283.295/pdf	English	null	THE SPACE OF WROCŁAW IN THE CONTEXT OF ECC 2016 MICROGRANTS PROGRAM REALIZATION – RESEARCH CONCLUSIONS	Journal of Education, Culture and Society	2,017	cc-by	6,504	Abstract In 2016, the title of the European Capital of Culture was awarded to Wrocãaw. To celebrate the year of 2016 citizens could partake in many cultural initiatives prepared specially for this occasion. The proposals aimed at the people of Wrocãaw included the ECC 2016 Microgrants program, under which any citizen could realise and gain funding, as well as obtain administrative and promotional support for their designed cultural activity. Field studies conducted during the Þ rst two editions of the program served as the basis for a report, which includes conclusions regarding the perception of the urban space submitted by task performers. Low-budget cultural activities proved to be an excellent pretext for the citizens to reconsider the issues of public space in Wrocãaw, access to cultural events or the manner of constructing ofÞ cial discourses about the city and its space. This article focuses on the issue of space in the context of Microgrants 2016 and presents the outcomes of the Þ eldwork. Key words: European Capital of Culture, space, culture, public space doi: 10.15503/jecs20172.283.295 doi: 10.15503/jecs20172.283.295 283 Journal of Education Culture and Society No. 2_2017 Agnieszka Wieszaczewska Faculty of History and Pedagogy, University of Wroclaw, Uniwersytecki Square 1, 50-137 Wrocãaw, Poland. E-mail address: agnieszka.wieszaczewska@uwr.edu.p Agnieszka Wieszaczewska Introduction – urban public spaces At the start of the Industrial Revolution, urban centres in Europe began to expand rapidly, and this process has continued to the present day. “The world is continually becoming more urban – the population in areas and estates classiÞ ed as urban is increasing. The growth rate is unprecedented. The rise in the number of megacities – cities a with populations over ten million – is a spectacular manifestation of modern urbanisation in terms of population concentration” (Grochowski, 2013, p. 9). It is not surprising, therefore, that cities get more attention from the press, in panel discussions and debates, regarding the manner of development of urban centres, and urban studies degree courses continue to be introduced into university cur- ricula. The cities themselves are facing new problems and the pressure of urbanization affects all the issues connected with life in the city, such as spatial, economic or environmental, as well as cultural and social aspects of 284 Local Cultures and Societies the city. How are we to stimulate the cultural development in cities? How can we make them accessible for everyone? How do we make cities more attractive for tourists through the available cultural activities? These are only some of the questions regarding the association of culture with the city. Nowadays, culture has become an integral and continuously more impor- tant factor in the development of large, medium-sized and small cities, as well as a “building material constituting the concepts of the urban, civic and European identity. Culture is crucial for proper functioning of larger com- munities” (Filar, & Kubicki, 2012, p. 135). ( p ) One of the most important issues of today’s urban planning is the role, con- dition and destination of public spaces in cities – both in metropolises and small centres. It is widely considered that moving away from the Athenian idea of the agora, where people could meet to freely exchange their concepts, organise and experience things together, is caused by economic changes, among other things. When comparing the contemporary city with an international corpora- tion, Richard Sennet demonstrated that what once was one of the essential fea- tures of urban centres – the opportunity for strangers to meet – has gradually started to disappear. And it is characteristic both of large, present-day capital- ist companies, as well as cities. Introduction – urban public spaces Sennett (2007) stated that: The sense of fraternity as a shared fate, a durable set of common interests, has been weakened. Socially, the short-term regime produces a paradox. People work intensely, under great pressure, but their relations to others remain curiously superÞ cial. This is not a world in which getting deeply involved with other people makes much sense in the long run. The American sociologist considers encounters not only as physical inti- macy, but, primarily, as an opportunity to establish relationships of varying intensities. The decline of the functions of traditional urban spaces is also connected to their gradual privatization, architectural procedures aimed at discouraging people from using them, or cultural changes (for instance, the presence of television in almost every household is considered by many the reason why people do not want to meet in a public space to establish relationships). p ) Public space should be distinguished from the city by a couple of features. First of all, it needs to be accessible to everyone – every citizen has the right to use it, as long as they are not breaking the code of conduct pertaining to such places, adopted by a given community. Streets, parks, squares and plazas are basic types of public spaces in cities. Apart from being accessible, a public space must also be authentic (meeting the real needs of citizens) and safe. A public space also needs to be “possible to grasp: by looking at it, taking a walk in it, by the possibility of bringing people, who can later be recognised and possibly identiÞ ed with, together (...) as well as through elusive thoughts and deeper reß ection” (Frysztacki, 2005, p. 152). p ( y p ) Cultural initiatives in the public space can be a factor sustaining the local communities occupying and enriching those places. Sometimes, these initia- 285 Journal of Education Culture and Society No. 2_2017 tives can lead to the establishment of new relationships. In the case of people living on the same street, estate or neighbourhood, where the only thing that the residents share is “the same address”, cities cannot be considered commu- nities. After all, a “community consists of people socially interacting and relat- ing to each other within a given area and having common interest or a sense of group and spatial identity as an element of common bonds” (Starosta, 1995, p. 31). “ECC 2016 Microgrants” Program as an Example of Limited Support for Cultural Initiatives The idea to include cultural heritage and tradition of particular European cities in the processes of intracontinental integration was articulated in the 1980s. The idea of Melina Mercouri, the then Minister of Culture of Greece, has been widely acknowledged and started to function under the name of the European City of Culture since the beginning of the new millennium. The belief that cultural heritage can support the continental bonding process had obviously surfaced previously. K. Kowalski, an ethnologist, mentions this con- cept in his work (2013): Notions from the analytic language of social studies appear in the institutional discourse of the EU. Along with heritage, it is culture, identity and social cohe- sion that play a big role in this discourse, since they have already preserved their descriptive and performative role and persuasive position. They became mobiliz- ing metaphors used to craft European culture, European identity and European awareness (p. 11). The European Capital of Culture program was launched in 2000. Its main idea is intra-European integration based on intercultural dialogue and bring- ing together diverse cultures, as well as promoting and maintaining Europe’s cultural heritage. The city chosen as the European Capital of Culture for the entire year becomes the cultural centre of the entire continent, and the place where miscellaneous cultural initiatives are held, which show the richness of the chosen Capital’s heritage. Edwin Bendyk (2012) questions the entire concept, by calling the competition for the title of the European Capital of Culture: (...) a strange initiative, [in which], cities (...) take part in an infantile venture – a contest, where they need to show off in front of a more or less arbitrarily chosen jury. And all this for what? To prove, just like schoolchildren, that they have done their homework when it comes to culture, and that they can be promoted to the next grade (p. 97). There are more people voicing their objections against the European Capi- tal of Culture, which does not change the fact that Polish cities dream of this title. Since 2008, the following cities have applied to participate in the contest: Biaãystok, Bydgoszcz, Gdaęsk, Katowice, Lublin, âódĮ, Poznaę, Toruę, Szc- zecin, Warsaw and Wrocãaw. In December 2008, Wrocãaw City Council decided that the city would take part in the European Capital of Culture 2016 contest. Introduction – urban public spaces When talking of a real local community, it turns out that spatial proximity is not as important as a certain type of closeness of its residents. Theobald & Siskar (2010) state that: The degree to which a particular place in fact constitutes a community depends not on the number of people living in close proximity to one another, but on the amount of life circumstances that are shared. A small number of ranch families spread out across vast spaces within a particular watershed may be more of a com- munity than a densely-populated suburb (p. 198). Charles Montgomery considers the lack of joint cultural and social initia- tives within the so-called scattered cities as one of the main reasons for citizens’ declarations regarding their low life satisfaction. According to his diagnosis, it is because of the Bauman’s “neighborhood without neighbors” concept, that Americans are continuously less satisÞ ed with their surroundings. Szatan (2012) explains that this term describes (...) people, who live near each other in close proximity, but remain isolated when it comes to other things, and do not know each other well. The lack of common interests minimizes the role of mutual commitments among people living in the same area (p. 100). However, Montgomery optimistically depicts how joint initiatives improved the situation of unsatisÞ ed residents in some expansive American suburbs. Their sense of security, the level of trust in neighbours and the general evaluation of their life situation have improved straight away (Montgomery, 2015). ) In this article I would like to outline the results of the research conducted by the University of Wrocãaw research team in 2014, concerning one of the cultural programs implemented as part of the European Capital of Culture 2016 initiative in Wrocãaw. The “ECC 2016 Microgrants” program aimed to increase the degree of citizen participation in the celebration of the year of 2016, as well as to encourage them to take part in the city’s cultural life, basi- cally to co-create it. The program also aimed to revitalize the public space in Wrocãaw by helping the citizens implement their own cultural initia- tives, for instance in their immediate surroundings (districts, estates, streets, or buildings). The conclusions presented concern one of the many issues addressed by the researchers, namely the people involved in the urban space narrative. 286 Local Cultures and Societies “ECC 2016 Microgrants” Program as an Example of Limited Support for Cultural Initiatives Throughout the follow- ing year, an interdisciplinary team worked on a social diagnosis which became the basis for creating the contest application. The preparation of the applica- tion, which is the basic document enabling the participation in the contest for the ECC title, began in Wrocãaw in February 2010. The document, bearing the slogan “Spaces for Beauty,” was forwarded to the Ministry of Culture and National Heritage six months later. In June 2011, Wrocãaw was announced as 287 Journal of Education Culture and Society No. 2_2017 Journal of Education Culture and Society No. 2_2017 the winner, and the ECC 2016 ofÞ ce responsible for the preparation of Wrocãaw for the celebration of the year of 2016 was set up a year later. “ECC 2016 Microgrants” is one of the European Capital of Culture 2016 celebration programs which aims to “include active residents of Wrocãaw and co-create Wrocãaw as part of the European Capital of Culture 2016 project by identifying, promoting and supporting social initiatives, as well as co-organ- ising initiatives Þ tting the idea and concept of the ECC 2016” (Regulations Regarding Acquiring Partners to the Project Co-Organising Program by the Impart 2016 Festival Centre). p ) The program organisers speciÞ ed its main goals. The program should: The program organisers speciÞ ed its main goals. The program should: • Refer to and comment on discussions being currently underway in Wrocãaw concerning bottom-up cultural initiatives, access to cul- ture, Wrocãaw’s local community issues (e.g. in estates) and the more common innovative animation and cultural initiatives, • Help and support the progress of cultural and social events initiated by residents, especially those concentrated far from the city centre (this concerns mostly the administrative and Þ nancial support), • Enliven the urban space, particularly by increasing the number of initi- atives realized by the citizens (not by non-governmental organisations, cultural institutions, etc.), • Act as one of the development mechanisms for permanent cooperation strategies between the citizens of Wrocãaw and cultural institutions, • Refer to the cultural participatory budget concept and the main mes- sage included in the Wrocãaw’s ECC 2016 contest application, namely the reinforcement of cultural participation understood in the broad sense, and counteracting exclusions from culture. “Microgrants ECC 2016” program has been realized since 2014, when two pilot editions of the program with a relatively low budget and a low number of Þ nanced projects were implemented. “ECC 2016 Microgrants” Program as an Example of Limited Support for Cultural Initiatives Their number substantially increased in the following year, along with the amount of funds allocated to this initiative (although the maximum amount for the project (PLN 5000), which is approxi- mately EUR 1200, remained the same since the program’s inception). From 2014, 11 calls for proposals were announced, within which over 100 projects were realised in different areas of the city and the voivodeship. Research methodology The goals of the evaluation project of ECC 2016 Microgrants were social and practical, as well as of a cognitive nature. The research procedure, along with the analysis of the materials collected, was held during the Þ rst two edi- tions of the program. The conclusions were used to improve the initiative in the subsequent planned stages. q p g The three main goals for the research team included examining the ECC 2016 Microgrants functioning mechanism in the context of Wrocãaw’s environ- 288 Local Cultures and Societies ment, answering the question whether the program had fulÞ lled its assump- tions, and Þ nally, developing a catalogue for future recommendations. The whole research process aimed at providing answers to the following questions: ment, answering the question whether the program had fulÞ lled its assump- tions, and Þ nally, developing a catalogue for future recommendations. The h l h d d h f ll y p g g whole research process aimed at providing answers to the following question 1. How is the initiative perceived by the diverse groups relevant to the program: the jury, the organizers, the applicants and the citizens of Wrocãaw? 2. How do the above-mentioned groups understand the critical concepts present in the Wrocãaw’s European Capital of Culture contest applica- tion, such as culture, beauty, participation? y p p 3. How can the program’s functioning be improved in its subsequent editions? 4. Did the program accomplish its particular goals, which were highlighted on the project’s home page by the organisers, during the realisation of the Þ nanced projects? 5. Were there any territorial regularities in relation to the contest applications? Research environment. The research included mostly people who were directly associated with the realization of the Þ rst two editions of the ECC 2016 Microgrants program, namely: Research environment. The research included mostly people who were directly associated with the realization of the Þ rst two editions of the ECC 2016 Microgrants program, namely: Microgrants program, namely: 1. Jurors who evaluated the submitted proposals for artistic and animation projects; j 2. Program’s beneÞ ciaries; 3. Individuals employed in the program’s implementation (those respon- sible for consulting about the program with interested potential beneÞ - ciaries, among others). The following documents have also been analysed: The following documents have also been analysed: 1. Wrocãaw’s winning contest application, which granted the city the title of ECC 2016. 2. Research methodology Rules of the ECC 2016 Microgrants program. 2. Rules of the ECC 2016 Microgrants program. 3. (“Regulations Regarding Acquiring Partners to the Project Co-Organis- ing Program by the Impart 2016 Festival Centre”). 4. The application form, as well the projects submitted in the Þ rst two edi- tions of the program which successfully passed the formal evaluation. 5. Statistics and studies regarding the Wrocãaw space, and most impor- tantly, its socio-cultural contexts, such as access to cultural institutions, the activity of non-governmental organisations and others. y g g The analysis of the above-mentioned documents aimed at providing answers to questions about the culture accessibility in different locations in Wrocãaw (the analysis was conducted mostly on the estate level) and the level of operations of cultural institutions and third sector organisations and their location in the city space. The analysis also focused on bottom-up activities, the language used in the program’s application form and the understanding of ECC 2016 keywords. 289 Journal of Education Culture and Society No. 2_2017 Journal of Education Culture and Society No. 2_2017 Research Structure Research Structure The research was split into three stages to guarantee constant veriÞ ca- tion and supplementation of the data already gained. Its objective was also to compare the evaluation results with the subsequent program’s calls for proposals, their assessment, and realisation of the initiatives which gained funding. In every stage of the research qualitative in-depth interviews were conducted with the program’s beneÞ ciaries, the jury and the employees of the Impart 2016 Festival Centre, who were responsible for the ECC 2016 Microgrants program. In the Þ rst stage, research material was collected during a focus group interview with the members of the selection board. The Þ rst stage of the research also included an analysis of the already existing data, while the second and the third incorporated a transparent observation during the realisation of the program’s Þ nanced projects. The last stage of the research featured an analysis of spatial distribution of the submitted and implemented initiatives. p It is worth noting that the project was fully commissioned and Þ nanced by the Impart 2016 Festival Centre. Research tools During the evaluation, the research team applied tools used in qualitative research, and tried to use diverse tools to provide a broad view of the phe- nomenon described herein. A poll, characteristic of quantitative studies, was included. Denzin and Lincoln (2010) justify the use of multiple methods in the following way: Triangulation, that is using a variety of methods, reß ects the attempt to further understand the examined phenomenon. (...) A combination of many methodologi- cal practices, empirical data, perspectives and observers in one research constitutes a strategy combining strictness, panache, complexity, richness and depth of every research (pp. 26-27). By using diverse research strategies and perspectives (the members of the research team represented different scientiÞ c disciplines, such as pedagogy, cultural anthropology, cultural studies and philosophy, among others) as broad a spectrum of the researched phenomena as possible was attempted to be obtained. The basic tools used in the research process included the analysis of the already existing data (qualitative and quantitative), qualitative interview, observation, cultural mapping and interview surveys. Researchers also conducted focus group interviews with experts involved in the evaluation of contest applications, and on the map of the city of Wrocãaw we have marked sites relevant to the Þ rst two editions of the ECC 2016 Microgrants program: sites where the projects were to be carried out (all those which were given a positive mark in the formal evaluation), as well as those which were granted funding. By doing this, the research team wanted to answer the question about the presence of regularities in relation to spatial distribution of initiatives. 290 Local Cultures and Societies Although the research project focused on several research questions, this article reports the conclusions which are related with the issue of space. The whole report is available online. Although the research project focused on several research questions, this article reports the conclusions which are related with the issue of space. The whole report is available online. Cultural mapping – ECOC 2016 Microgrants in the Wrocław space The method of cultural mapping is integrally connected with engagement of the community in creating its cultural area. Turning attention on Þ rst edi- tion of ECOC 2016 Microgrants in Wrocãaw in the context of culture mapping helps to create a Wrocãaw map with selected categories of places important from the perspective of the whole project analysis. Due to limited time and type of research cultural mapping method was an inspiration for us; how- ever, some of its aspects were not applied to the research. Cultural mapping engages society which identiÞ es and documents local cultural resources. While researching we identiÞ ed such elements of culture as: galleries, workshops, local events, but also memories, personal stories, values. (…) The crucial aim of this mapping is to help society identify (…) cultural diversity for regional economic and social development. The whole research aimed to identify “places of power” in Wrocãaw understood as places with culture-producing potential, based also on bot- tom-up initiatives of citizens. Including in a map such vital points as culture houses, cinemas, theaters, etc. aimed at understanding whether the existence of this type of project affects cultural initiatives of citizens. The image of citi- zens’ activity in the Microgrants program context has primarily diagnostic value, focusing on two most important issues: 1. Map as a basis to plan future lines of action for urban cultural policy. 2. Map as an identiÞ cation of places with rich cultural offerings and those with a deÞ cit. Were citizens from districts with developed infrastructure more active in seeking support for their micro grants ideas? A supplement for the map is a qualitative analysis of interviews focusing on how to value an urban space by beneÞ ciaries in the Þ rst and second recruit- ment of the project. The readers will Þ nd it in a following paragraph of this article. Cultural mapping helped to illustrate the implementation of the Micro- grants program in urban space and the information gathered became a sup- plement for the Þ nal study report. A study is a graphic presentation of the following data locations: g 1. Location of all upcoming events submitted to the program, 2. Locations of events selected by the committee for implementation, y 3. Location of existing cultural institutions, NGOs, etc., 4. Location of Wrocãaw social problems on a district level. Cultural mapping – ECOC 2016 Microgrants in the Wrocław space Information was gathered through the analysis of statistical data and publications on this topic (Kãopot, & Skiba, 2006). 291 Journal of Education Culture and Society No. 2_2017 Journal of Education Culture and Society No. 2_2017 As a result of inquiry a map of Wrocãaw was created. It is a starting point in discussions about civic participation, culture-producing role of cultural institution and NGO’s as well as the ‘cultural’ condition of particular Wrocãaw districts. This can inß uence municipal policy on culture or programs which may strengthen and activate citizens. The Þ nal report includes the map with a key. The study on a spatial aspect of micro grants program emphasized a serious issue. The vast majority of projects submitted to the program were located in the very centre of the city. There was not a signiÞ cant relevance between the existing cultural institutions or NGO’s and the number of submit- ted applications. The dependency found during the analysis was simple: the closer to the city centre, the more applications were submitted. The exception was Nadodrze district with signiÞ cant amount of applications. The Nadodrze district is currently under the process of revitalisation. As a result of this a lot of NGO’s are located there, which is the very probable explanation for the increased number of applications. The very interesting point was that for some jurors the distant localisation of the proposed project was a reason for higher rating of the application. It is also visible in the map that although the majority of the projects were supposed to happen in the city centre, those selected for realisation were located in the more distant districts. It means that Microgrants can become not only a valuable way for diagnosing social demand on cultural offer in a city, but also an inspiration for citizens to set up some cultural activity in their neighborhood. Two narratives about sites – recovering the urban space in action And it really upsets me that, as a mom, I have to go to the city centre in order to show anything to my kid. The same interviewee also raised the issue that when a particular institu- tion organises cultural events, most of them are closed initiatives (e.g. activ- ities in schools addressed only to the students of a given facility). In a con- versation with the performer of the project in question (“Ready to START”), another important issue concerning the contemporary image of public space has surfaced, namely the low degree of citizens’ integration, not only in the same estates, but also of close neighbours: “Because there is nothing happen- ing, these people do not participate in the life on estates. They take care of eve- rything… shopping, school in some sites. Even work. There is nothing going on to enable the people to meet somewhere.” p p The implementation of the project has resulted not only in a series of events and activities for the residents of Ksičİe Maãe, but also in a formation of a work- ing group ready to further improve the estate. During the research, only one of the beneÞ ciaries had voiced her reservations about the poor cultural offer in her immediate surroundings in a clear manner. A juror, who considered the fact that a given project was to be realised outside the city centre a relevant factor in the evaluation of applications, also commented on the disproportion in the access to city‘s cultural resources. “For me, the geographical indicator was also signiÞ cant, although it was not a directly speciÞ ed criterion. How- ever, I rated higher projects from far-off estates, or held in estates lacking cul- tural activities, which does not necessarily mean peripheral estates. The estates of PrzedmieĤcie Oãawskie, Huby, Pawãowice, and Ksičİe Maãe are really far from the city centre and it caught my attention right away.” y g y g y Initiatives located in areas of Wrocãaw considered inferior, such as Nadodrze and PrzedmieĤcie Oãawskie, have also been Þ nanced. The applicants did not hide the fact that this issue was important to them: “Well, we live in Nadodrze, and it is safe to say that this district is not the richest, so it is a chance for us to get ahead. Two narratives about sites – recovering the urban space in action (all quotes used in this subsection come from the interviews carried out during the research) One of the most problematic areas within the research and a reß ection regarding the evaluation was urban space treated as a theme, a protagonist and a medium in the ECC 2016 Microgrants program. Indicating the sites of implementing the proposed initiatives on the map (both those which gained funding, and those not realised) from the Þ rst and second call for proposals was a starting point for the analyses. The issue of space has also surfaced in applications and interviews with the beneÞ ciaries, the contest’s jurors and employees of the ECC 2016 centre. It was very interesting hearing them convey their thoughts about the city, the needs of its citizens and the role of space in realisation of the program’s main idea: spaces for beauty. The respondents very often mentioned the issue of access (or lack thereof) to the infrastructure used by the citizens of Wrocãaw in leisure activities. One of the interlocutors clearly stated that the restricted access to culture on offer on her estate was a motivation for her to partake in this contest. The beneÞ ciary wanted to “take matters into her own hands,” also because she believed this situation hurt mostly children and parents: 292 Local Cultures and Societies Local Cultures and Societies Local Cultures and Societies I felt an internal need. (...) I work in Ksičİe [one of Wrocãaw’s estates - A.W.] and I have been living there for twelve years. I am also the manager of this library, so in a sense I am a public Þ gure and I observe such things. In the meantime, I have also become a mom. Some of my private needs, were kind of… I did not notice those things. And it really upsets me that, as a mom, I have to go to the city centre in order to show anything to my kid. I felt an internal need. (...) I work in Ksičİe [one of Wrocãaw’s estates - A.W.] and I have been living there for twelve years. I am also the manager of this library, so in a sense I am a public Þ gure and I observe such things. In the meantime, I have also become a mom. Some of my private needs, were kind of… I did not notice those things. Two narratives about sites – recovering the urban space in action I think that in the context of developing culture and supporting bottom-up initiatives it is very important.” y Nadodrze is a peculiar estate because of its revitalization. The emphasis placed on artistic and social growth of the estate has been noticed by the ben- eÞ ciary and the realisation of the microgrant Þ tted perfectly with the initiatives undertaken for years by various institutions and non-governmental organisa- tions. The other place which evokes bad associations in most of the citizens of Wrocãaw is PrzedmieĤcie Oãawskie, also known as the “Bermuda Triangle”: “The Bermuda Triangle (...) is the most neglected estate in Wrocãaw. Everyone focuses on Nadodrze, and yet nobody mentions the «Triangle». That is why we wanted to concentrate on PrzedmieĤcie Oãawskie, which holds many remnants of the past, such as stories and buildings.” 293 Journal of Education Culture and Society No. 2_2017 Journal of Education Culture and Society No. 2_2017 However, most of the awarded projects in the Þ rst two editions of the pro- gram were carried out across an area larger than an estate, and often involved the entire city, or even the Lower Silesia region. In terms of location, we have observed another interesting regularity connected to the ECC 2016 Micro- grants, apart from the strong focus on close “neighbourhood,” which charac- terized some of the projects and which was perceived by the research team as an enormous advantage. It was the attempt of alternative urban space story- telling in the realised projects. The beneÞ ciaries spoke of their ideas to create an alternative to the ofÞ cial discourse on the space of Wrocãaw, which is no longer just a dynamic, European metropolis and the famous “meeting place.” The goal of many initiatives was showing the city from a lesser known side and accentuating the beauty of areas that are not promoted as worth seeing: “our postcards do not show any of the known places. If it is the Wrocãaw of today, it would not be the Main Square or Old Town because there are a lot of places which depict [Wrocãaw] in a prettier and more genuine way.” A similar reß ec- tion was shared by the applicant of a project covering the entire Lower Silesia, which involved taking photographs of unknown and forgotten monuments of the voivodeship not included in tourist guides. Two narratives about sites – recovering the urban space in action Apart from accentuating the beauty of such places, the respondents pointed to another strategy of creating alternative stories about the city, namely giving voice to the citizens. One of the projects encouraged people connected to a particular estate to come up with a story about it, which would be later permanently exhibited in the public space. Another project focused on creating a collective diary of people of Wrocãaw. In the spatial dimension, the ECC 2016 Microgrants pro- gram has emphasised two important issues. First of all, they can become a val- uable way of diagnosing social demand for the cultural events realized by the city. In order to achieve that, all the submitted applications would need to be accordingly analysed (What needs were reported by applicants? Are there any estates that are diagnosed by the citizens as lacking in these kinds of cultural events?). Secondly, the microgrants can actually realise the “spaces for beauty” slogan, under which the Wrocãaw ECC contest application was prepared. According to the application, beauty is multidimensional and created by the human energy Þ lling the space and resulting from interpersonal relations. Cre- ating places for beauty requires the involvement of citizens who perceive space in their own individual way. From the interviews with participants it can be deducted that the perception of Wrocãaw’s spaces is oftentimes different from the city’s ofÞ cial presentation – a facade of monuments and great events – and this sensitivity to the forgotten urban space is the main focus of many of the initiator’s contest projects. p j The research team developed the catalogue of recommendations for future editions of the program. The recommendation concerned funding, methods of recruitment, how the competition documents should be constructed. When it comes to space, the basic recommendation was stronger emphasis on Micro- grants sub local character. Applications are small and are often territorially 294 Local Cultures and Societies limited. This feature can be turned into value – and it can be treated as a means to activate local societies at a neighborhood level (or different, not covering the entire city). y) The research team recommend to integrate cultural activities within the Microgrants – on the basis of territorial aspects – with other activities taken by public institutions and non-governmental organizations in given Wrocãaw districts. Two narratives about sites – recovering the urban space in action Institutional and political context is favorable: Wrocãaw faces chal- lenge i.e., on the one hand, the future of district committees, and on the other hand – growing popularity of the Wrocãaw Participatory Budget. Research- ers recommend to deepen sub local identities – with particular consideration to their activities in culture, politics or education. Instead, the research team discourage municipal government actions which can weaken sub local identi- ties to unite Wrocãaw identity.At an operational level – managing Microgrants program should be: • More territorially diversiÞ ed. BeneÞ ciaries’ service points and meeting points dedicated to the program should be localized in neighborhoods. • In a similar way one can think of channels promoting the program: e.g. posters informing about micro grants could be at schools, parishes, seniors‘ clubs, shops, universities, culture houses and student clubs. In such places – and even, having calm weather conditions – one can organize public meetings informing about the program. Conclusions The activities depicted in this article were related to the celebration of the European Capital of Culture 2016 in Wrocãaw. Small-scale funding of citizen’s cultural activities is a complete novelty here, although initiatives of this type are becoming more and more common in Poland. Low-budget, local activities of groups of people associated with a given place in a city can be viewed as the resistance shown by the people of Wrocãaw to the progressive uniÞ cation of cultural endeavours, as well as the prevailing manner of understanding what culture participation is (that is of being a passive observer of events offered by various cultural institutions). Microgrants have encouraged the citizens to do something together with their neighbours, revitalize their estates’ public spaces, and sometimes to even create one. They have also enabled overcom- ing of the ofÞ cial, promotional and touristic discourse about a city in which there was no place for spaces close to the project’s performers. The initiatives described fully Þ t in with the trend of “new localism,” which has been described as “educational, social and cultural activities taking place completely in a spe- ciÞ c location, with the aim of gradual recovery of the place’s signiÞ cance and peculiarity in spite of progressing globalisation” (Gruenewald, & Smith, 2010, p. xiii). Such initiatives have strong educational potential and let people who do not partake in the city’s cultural life take responsibility for “their” piece of the occupied space, since these initiatives teach self-agency and encourage 295 Journal of Education Culture and Society No. 2_2017 Journal of Education Culture and Society No. 2_2017 Journal of Education Culture and Society No. 2_2017 people to have a real inß uence on the inhabited area. people to have a real inß uence on the inhabited area. References 1. Denzin, N. K., & Lincoln, Y. S. (2010). Metody badaę jakoĤciowych 1 [Qualitative research methods]. Warszawa: Wydawnictwo Naukowe PWN. 2. Frysztacki, K. (2005). Mičdzy przestrzeniĈ i publicznoĤciĈ miejskĈ [Between the space and the city public]. In: A. Wallis, B. Jaãowiecki, A. Majer, & M. S. Szczepaę ski (Eds.), Przemiany miasta: Wokó ã socjologii Aleksandra Wallisa [Transformation on the city. Around the sociology of Alexander Wallis] (pp. 151-158). Warszawa: Wydawnictwo Naukowe Scholar. j g y Alexander Wallis] (pp. 151-158). Warszawa: Wydawnictwo Naukowe Scholar. 3. Grochowski, M. (2013). Wstčp do wydania polskiego. In: Ch. Landry. (Ed.), Kreatywne miasto. Zestaw narzčdzi dla miejskich innowatorów [Creative city. Toolkit for urban innovators] (pp. 9-22). Warszawa: Narodowe Centrum Kultury. y 4. Gruenwald. D. A., & Smith, G. A. (2010). Place-Based Education in the Global Age. New York, London: Routledge. Taylor & Francis Group. g y p 5. Kãopot S. W., & Skiba W., (2006). Atlas Spoãecznych Problemów Wrocãawia [The Atlas of Wrocãa- w’s Social Problems]. Wrocãaw: Silesia. 6. Kowalski, K. (2013). O istocie dziedzictwa kulturowego – rozwaİania [Abouth the essence of cul- tural heritage – considerations]. Kraków: Mičdzynarodowe Centrum Kultury. 7. Filar P., & Kubicki P. (Eds.). (2012). Miasto w dziaãaniu. Zrównowaİony rozwój z perspektywy oddolnej [City in action. Sustainable develompnet from the bottom-up perspective]. War- szawa: Instytut Obywatelski. y y 8. Montgomery, Ch. (2015). Miasto szczčĤliwe. Jak zmieniaþ nasze İycie, zmieniajĈc nasze miasta [Good city. How to change our lives by changing our cities]. Kraków: Wysoki Zamek. 9. Sennett, R. (2008). Elastyczne miasto obcych sobie osób [Flexible city of strangers]. Le Monde diplomatique. Miesičcznik spoãeczno-polityczny, 4. Retrieved from http://monde-diplomatique. pl/LMD25/index.php?id=9. p p p 10. Starosta, P., (1995). Poza metropoliĈ. Wiejskie i maãomiasteczkowe zbiorowoĤci lokalne a wzory porzĈdku makrospoãecznego [Beyond the metropolis. Rural and small-town local communities and patters of macro-social order]. âódĮ: Wydawnictwo Uniwersytetu âódzkiego. p ] y y g 11. Szatan, M. (2012). Zanikanie przestrzeni publicznej we wspóãczesnych miastach [The eva scence of public space in modern cities]. Palipsest. Czasopismo socjologiczne, 2, 91-102. 12. Theobald, P., & Siskar, J. (2010). Place: Where Diversity and Community Can Converge. In: D. A. Gruenwald, & G. A. Smith (Eds.), Place-Based Education in the Global Age (pp.197-220). New York, London: Routledge. Taylor & Francis Group.
https://openalex.org/W4249813170	https://www.qeios.com/read/N1AMQR/pdf	English	null	GAIA Level 1 Neonatal Death in a Non-viable Live Birth	Definitions	2,020	cc-by	139	Open Peer Review on Qeios Open Peer Review on Qeios GAIA Level 1 Neonatal Death in a Non- i bl Li Bi th National Cancer Institute Qeios ID: N1AMQR · https://doi.org/10.32388/N1AMQR Qeios · Definition, February 2, 2020 Open Peer Review on Qeios Source National Cancer Institute. GAIA Level 1 Neonatal Death in a Non-viable Live Birth. NCI Thesaurus. Code C127969. Thesaurus. Code C127969. GAIA Level 1 Neonatal Death in a Non-viable Live Birth is defined by three criteria: first, the infant must be live born; second, at least one of the following requirements must be met: a) Gestational age less than 22 weeks (a GA level of certainty of one); OR b) Birth weight less than 500g; third, the death of the infant within the first 28 days of life must be documented. Qeios ID: N1AMQR · https://doi.org/10.32388/N1AMQR 1/1
W4210659354.txt	https://link.springer.com/content/pdf/10.1007/s10439-022-02921-4.pdf	en		Biomechanical Markers of Forward Hop-Landing After ACL-Reconstruction: A Pattern Recognition Approach	Annals of biomedical engineering	2,022	cc-by	8,730	BIOMEDICAL ENGINEERING SOCIETY Annals of Biomedical Engineering, Vol. 50, No. 3, March 2022 ( 2022) pp. 330–342 https://doi.org/10.1007/s10439-022-02921-4 Original Article Biomechanical Markers of Forward Hop-Landing After ACL-Reconstruction: A Pattern Recognition Approach PRASANNA SRITHARAN ,1 MARIO A. MUÑOZ ,2 PETER PIVONKA ,3 ADAM L. BRYANT,4 HOSSEIN MOKHTARZADEH ,5 and LUKE G. PERRATON 6 1 La Trobe Sports and Exercise Medicine Research Centre, La Trobe University, Bundoora, Australia; 2School of Mathematics & Statistics, University of Melbourne, Melbourne, Australia; 3School of Mechanical, Medical & Process Engineering, Queensland University of Technology, Brisbane, Australia; 4Centre for Health, Exercise and Sports Medicine, University of Melbourne, Melbourne, Australia; 5Department of Mechanical Engineering, University of Melbourne, Melbourne, Australia; and 6 Department of Physiotherapy, Monash University, Melbourne, Australia (Received 11 October 2021; accepted 4 January 2022; published online 31 January 2022) Associate Editor Michael R. Torry oversaw the review of this article. Keywords—Anterior cruciate ligament, Feature selection, Principal component analysis, Knee osteoarthritis, Musculoskeletal modelling, Machine learning. Abstract—Biomechanical changes after anterior cruciate ligament reconstruction (ACLR) may be detrimental to long-term knee-joint health. We used pattern recognition to characterise biomechanical differences during the landing phase of a single-leg forward hop after ACLR. Experimental data from 66 individuals 12-24 months post-ACLR (28.2 ± 6.3 years) and 32 controls (25.2 ± 4.8 years old) were input into a musculoskeletal modelling pipeline to calculate joint angles, joint moments and muscle forces. These waveforms were transformed into principal components (features), and input into a pattern recognition pipeline, which found 10 main distinguishing features (and 8 associated features) between ACLR and control landing biomechanics at signiﬁcance a ¼ 0:05. Our process identiﬁed known biomechanical characteristics post-ACLR: smaller knee ﬂexion angle; less knee extensor moment; lower vasti, rectus femoris and hamstrings forces. Importantly, we found more novel and less well-understood adaptations: smaller ankle plantar ﬂexor moment; lower soleus forces; and altered patterns of knee rotation angle, hip rotator moment and knee abduction moment. Crucially, we identiﬁed, with high certainty, subtle aberrations indicating landing instability in the ACLR group for: knee ﬂexion and internal rotation angles and moments; hip rotation angles and moments; and lumbar rotator and bending moments. Our ﬁndings may beneﬁt rehabilitation and assessment for return-to-sport 12– 24 months post-ACLR. INTRODUCTION The single-leg hop-for-distance is routinely used in the evaluation of individuals after surgery to reconstruct a ruptured anterior cruciate ligament26,31 (ACL). During the landing phase, the individual must arrest the forward motion of the body, while supporting the body against gravity.38 Due to the dynamic nature of the task, and the consequent neuromuscular demands at the knee, the landing phase of single-leg hop-forlanding is known to stress the ACL1. During single-leg landing tasks, ACL-reconstructed (ACLR) individuals have shown altered biomechanics at the hip, knee and ankle12,15,25,27,45 when landing on the involved knee. In particular, ACLR individuals have demonstrated smaller peak knee ﬂexion angles20,27,28,38,45 and smaller peak knee extensor moments12,20,27,28,38 compared to controls and/or the uninjured limb. However, these ﬁndings are based on discrete data points in the landing phase determined a priori, and do not take into account the whole temporal waveforms of these biomechanical variables.4 Computer-based musculoskeletal modelling is a powerful, non-invasive approach29 that can facilitate an estimation of altered biomechanical control strategies after ACLR for many types of movements. Address correspondence to Prasanna Sritharan, La Trobe Sports and Exercise Medicine Research Centre, La Trobe University, Bundoora, Australia. Electronic mail: P.Sritharan@latrobe.edu.au Prasanna Sritharan and Mario A. Muñoz are co-ﬁrst authors. 330 0090-6964/22/0300-0330/0 2022 The Author(s) Hop-Landing Biomechanics After ACL-Reconstruction However, the data resulting from these analyses is multidimensional and demonstrates a non-linear relationship with quasi-periodic temporal dependence.3,8 Furthermore, the data exhibits variability, even within subjects and sessions3,6. This variability can be attributed to measurement error (e.g. inconsistent marker alignment and instrumentation3,6) and/or the individuals’ neuromuscular adaptations to their impairments6. The latter often results in functionally optimal, although abnormal, movement patterns. Movement variability complicates the objective examination of the data. In this regard, pattern recognition techniques may be suitable for the examination of biomechanical waveform data with such inherent variability. Principal Component Analysis (PCA) is one of the most commonly used approaches for analysing variance in multivariate data, and can be applied to the analysis of biomechanical temporal waveform data using pattern recognition methods.6 PCA can reveal clinically relevant information that would otherwise be difﬁcult to interpret from the original waveforms in healthy5,16,34,44 and pathological8,18,21,23,33,35 movement patterns. PCA attempts to ﬁnd a smaller set of new, non-redundant features, known as principal components, which sufﬁciently capture the observed total variation in the original variables3. It is unbiased and it does not require an a priori determination of features to extract.5 Although principal components are mathematically-abstract features, meaningful interpretation of the principal components can be undertaken with expert knowledge of human motion.3 Often, PCA is followed by statistical hypothesis testing,5,8,44 classiﬁcation2,16 or regression models21 to identify those features that are good predictors of the presence of anomalous movement patterns. Despite the widespread use of PCA in biomechanical studies, only two studies have used PCA to investigate diﬀerences in biomechanics between ACLR individuals and controls during dynamic tasks. Both Leporace et al.21 and Sanford et al.35 investigated walking gait post-ACLR using PCA and post-hoc classiﬁcation methods; however, these studies only examined biomechanical variables associated with the knee. Leporace et al.21 examined only knee-joint angles, ﬁnding that aberrations in knee internal rotation and knee adduction angles during gait were more important than knee ﬂexion angle in distinguishing between ACLR and controls. Sanford et al.35 additionally found that the knee adduction moment, which was elevated in early and late stance, was also a key variable in distinguishing between the groups. However, neither study included variables at the trunk, hip or ankle, and did not consider muscle forces. To our knowledge, no study using pattern recognition methods has examined a demanding dynamic task that is 331 known to stress the ACL, such as the single-leg hopfor-distance. Understanding the key distinguishing features of single-leg hop-for-distance may help improve the clinical assessment of individuals post-ACLR, and help mitigate re-injury or future osteoarthritis. Furthermore, it may, in future, facilitate the automated machine-based evaluation of individuals post-ACLR by interrogating subtle or hidden features of landing biomechanics that may be diﬃcult to observe directly. Therefore, the aim of this study was to apply musculoskeletal modelling and pattern recognition methods, to ﬁnd a minimum set of features of muscle forces, joint angles and joint moments, that could distinguish between the biomechanics of ACLR individuals and uninjured controls during a single-leg forward hoplanding task. As lower peak knee ﬂexion angle and knee extensor moment are distinct identifying characteristics of single-leg landing tasks post-ACLR38, we hypothesised that principal components associated with these respective variables would be included in the set of selected features. MATERIALS AND METHODS Data Collection and Musculoskeletal Modelling Sixty-six participants (28.2 ± 6.3 years, 24 (36%) women, height: 1.75 ± 0.10 m, mass: 78.3 ± 14.8 kg, 17 ± 3 months after ACLR) from a previously-described cohort of 111 eligible patients with unilateral single-bundle ACLR using a semitendinosis-gracilis tendon graft.31 and 32 uninjured control subjects (25.2 ± 4.8 years old, 17 (47%) women, height: 1.70 ± 0.08 m, mass: 68.0 ± 11.4 kg) participated in this study after providing informed consent. Ethical approval (ID: 1136167) for the study was provided by the Behavioural and Social Sciences Human Ethics sub-committee at the University of Melbourne. Surgery was performed by one of two experienced orthopaedic surgeons using identical surgical techniques. Inclusion criteria were a successful unilateral ACLR as determined by clinical examination by the orthopaedic surgeon. Subjects with self-reported knee instability, knee instability on clinical examination, revision ACLR, other surgery since ACLR or any other conditions affecting walking, sports activity or daily function were excluded. Data collection was undertaken at the Movement Research Laboratory, Centre for Health, Exercise and Sports Medicine, University of Melbourne. Participants completed an anticipated single-limb forward hop. The experimental procedure has been previously detailed,38 and only brieﬂy described below. Hop disBIOMEDICAL ENGINEERING SOCIETY 332 SRITHARAN et al. tance was normalised to 100% of leg length (greater trochanter to ﬂoor), with white tape used to mark the subject-speciﬁc take-off point on the ﬂoor and the landing point in the centre of the force plate. Participants took three steps forward using their preferred step length, took off at the marked take-off point, and landed on the cross in the centre of the force plate with the same leg with arms folded across their chest. ACLR participants used their affected leg while controls performed the task on their right leg. Landings were deemed successful if participants were able to maintain balance for ﬁve seconds after landing without moving their arms. Spatial marker trajectories were recorded using a 14-camera Vicon motion analysis system (Oxford Metrics, Oxford, UK) at 120 Hz and ground force data were recorded using a single force plate (AMTI Watertown, MA, USA). Modelling and analyses were performed using OpenSim 4.17 via the API using MATLAB R2020b (MathWorks Inc. Natick, MA, USA). For each participant, a musculoskeletal model was generated by scaling a generic 27-degree-of-freedom 92-muscle model. The ankles and subtalar-joints were modelled as pin-joints, while the hips and knees were modelled as a ball-joints. The metatarsophalangeal-joints were modelled as pin-joints, but locked at their reference positions. The head, arms and torso were merged into a single body which articulated with the pelvis via the back-joint, modelled as a ball-joint. For each participant and trial, joint angles were calculated using an inverse kinematics analysis by minimising the distance between model and experimental marker trajectories.24 Joint moments were calculated using inverse dynamics by applying the joint kinematics and measured ground forces to the model. Computed Muscle Control41 (CMC) was then used to calculate muscle forces. The internal knee adduction and rotation moments were excluded from CMC. Prior to calculation of muscles forces, the model’s torso centre-of-mass and trial joint kinematics were adjusted using the Residual Reduction Algorithm (RRA) to minimise dynamic inconsistencies. All analyses were performed for the landing phase of the task, deﬁned as the period from initial foot strike to maximum knee ﬂexion angle. Pattern Recognition Pipeline The temporal waveforms of 30 biomechanical variables (13 joint angles, 10 joint moments and 7 muscle forces) for each of 452 recorded trials from all subjects (301 ACLR trials; 151 control trials) were input into a pattern recognition pipeline, implemented in MA- BIOMEDICAL ENGINEERING SOCIETY TLAB, that found a minimal set of features, called principal components, that could best diﬀerentiate between the biomechanics of ACLR and control groups. Principal Component Analysis For each variable, the individual trial waveform data were ﬁrst transformed into a set of features called principal components that explain the maximum amount of variance in the original variables in a process known as Principal Component Analysis (PCA). This process is described in detail by Wrigley et al.,44 therefore we present only a summary here. PCA uses an orthogonal transformation that converts the n p matrix of waveform data X into an n p matrix of mutually uncorrelated principal component scores Z. There is an independent matrix X for each biomechanical variable, e.g. knee adduction angle or rectus femoris muscle force. For each variable’s matrix X, n rows represent trials, while p columns represent individual temporal samples of the waveform. ACLR and control trials are pooled such that n ¼ 452, and p ¼ 101, with each temporal sample representing 1% of the landing phase within the 0%-100% range. The principal component scores matrix Z for that variable was then calculated by the linear transformation: Z ¼ XU ð1Þ where U is the p p matrix of eigenvectors of R, which is the weighted correlation matrix of X. Each eigenvector uj , where j 2 ½1; p, in the matrix of eigenvectors U, is a set of coefﬁcients that is applied to each row of X to produce the matrix of principal scores Z. For each given biomechanical variable, every element zij of its matrix Z is known as a principal component score, and is a measure of the degree to which the shape of that variable’s waveform for trial i corresponds to the shape of the principal component eigenvector uj . All trials from all subjects were input into the PCA, however some participants recorded more trials than others. Thus, a normalized weight was applied to each row i 2 ½1; n in X when constructing the weighed correlation matrix R. Suppose row i represents a trial from participant S, who recorded qS trials in total. We deﬁne a unique factor: si ¼ 1 qS ð2Þ Therefore, the normalised weight applied to row i is: si wi ¼ Pn ð3Þ m¼1 sm Hop-Landing Biomechanics After ACL-Reconstruction Parallel Analysis For each biomechanical variable, the number of principal components was then reduced using Parallel Analysis (PA).13 Only those principal components that explained most of the variance in the data are retained, discarding the remainder. That is, if most of the variance in the data is explained by the ﬁrst k principal components, the remaining p k components were dropped. PA assumes that non-trivial principal components should have eigenvalues larger than principal components derived from random data with the same sample size and number of variables.10 To apply PA in the present context, N matrixes of size n p were generated, whose elements were independent and identically distributed random variables from a standard normal distribution. As larger N improves the accuracy of PA, we set N ¼ 1000. The n n correlation matrix was calculated from each random matrix, and its eigenvalues were extracted. The 95thpercentile of each eigenvalue from the random set was estimated and compared with the corresponding eigenvalues of R. Principal components with eigenvalues greater than the 95th-percentile were retained. After PA, 108 principal components were retained, a large number that may not have represented the most relevant modes of variation present in the data for the discrimination task.34 Weiss-Indurkhya Independent Features Selection To further reduce the number of retained features after PA, the Weiss-Indurkhya Independent Features Selection method43 was applied to the remaining principal components. For each retained variable, the mean principal component scores were calculated for the ACLR and controls groups, and compared using Welch’s t-test. Only features for which the betweengroup comparison returned t 2:0 were retained. For n ¼ 108, this is approximately a signiﬁcance level of 95%, i.e. a ¼ 0:05. Forty-six principal components were retained after this step. 333 because of its simplicity, effectiveness and short training time.32 As each iteration of SFS could produce different results due to the random nature of the 10fold cross validation, 1000 iterations of the SFS were performed, with only the 10 most-frequently selected principal components were retained. These ﬁnal 10 principal components were deﬁned as main features. Between-group differences in principal component scores for each of the main features were evaluated using Welch’s t-tests at a tighter signiﬁcance level of 99.9% (a ¼ 0:001), as these features were already signiﬁcantly different at the 95% level. Effect sizes were calculated using Hedges’ g-score. As principal components are abstract constructs, they require interpretation to have contextual meaning. For each feature, this was accomplished by: comparing the waveforms of the pooled original data that correspond to high and low principal component scores respectively8,36; the shape of the eigenvectors, i.e. waveform of the principal component coefﬁcients36; and the variance of the original data explained by that feature, i.e., the squared correlation between the temporal samples of the original data and the principal components.44 Associated Features SFS tends to exclude features that are well-correlated with the main features if they do not increase the accuracy of the model. These associated features can aid in interpretation of the results. For each main feature, we calculated the Pearson correlations q between it and every other remaining principal component after PA. Associated features were deﬁned as those remaining principal components which correlated moderately (0:5 jqj<0:7) or strongly (jqj 0:7) with their respective main feature at significance level a ¼ 0:05. Between-group differences in principal component scores for each of the associated features were evaluated using t-tests at signiﬁcance level t 2:0 for consistency with our Weiss-Indurkhya Independent Features Selection step, noting those which were also signiﬁcant at a ¼ 0:001. Sequential Feature Selection Sequential Feature Selection14 (SFS) was then used to obtain the ﬁnal minimal set of features from those 46 retained thus far. In SFS, the principal components were sequentially added to an empty candidate set until the addition of further features did not improve the stopping criterion or the number of valid features was exceeded. The stopping criterion was deﬁned as the 10fold cross-validated misclassiﬁcation rate of a Naı̈ve Bayes classiﬁer, with maximum number of valid features set to 10. The Naı̈ve Bayes Classiﬁer was used RESULTS For each variable retained after PA, median of 4 principal components accounting for an average of 98.5% of the total variance was retained (Table 1). This was reduced to 10 main features (Table 2) comprising 3 principal components of muscle forces (Fig. 1), 4 of joint angles (Fig. 2), and 3 of joint moments (Fig. 3). Seven features were able to distinguish BIOMEDICAL ENGINEERING SOCIETY 334 SRITHARAN et al. TABLE 1. Proportion of variance explained by each principal component retained after Parallel Analysis for the pooled data, followed by Weiss-Indurkhya independent feature selection and, finally, Sequential Feature Selection. Proportion of variance explained (%) Variable Muscle forces FGMAX FGMED FHAMS FRF FVAS FGAS FSOL Joint angles hHIPFLEX hHIPADD hHIPROT hKNEEFLEX hKNEEROT hKNEEADD hANKLEDF hPELVISTILT hPELVISLIST hPELVISROT hLUMBAREXT hLUMBARBEND hLUMBARROT Joint moments MHIPFLEX MHIPADD MHIPROT MKNEEFLEX MKNEEROT MKNEEADD MANKLEDF MLUMBAREXT MLUMBARBEND MLUMBARROT Description PC1 PC2 PC3 PC4 Gluteus maximus Gluteus medius Hamstrings Rectus femoris Vasti Gastrocnemius Soleus 76.5 58.4 73.2 68.5 64.1 65.3 77.9 9.4 26.5 15.0 15.4 17.4 20.4 15.6 6.4 9.9 6.3 8.3 7.4 7.7 3.5 4.8 3.0 2.6 5.1 4.0 3.0 Hip flexion Hip adduction Hip internal rotation Knee flexion Knee internal rotation Knee adduction Ankle dorsiflexion Pelvis tilt Pelvis list Pelvis internal rotation Lumbar extension Lumbar bending Lumbar internal rotation 95.0 87.4 89.3 93.3 82.9 93.4 51.2 95.8 86.8 93.0 96.5 86.1 86.8 3.7 10.2 8.9 4.8 13.0 4.9 36.5 3.5 10.1 6.1 2.9 10.7 10.2 Hip flexor Hip adductor Hip internal rotator Knee flexor Knee internal rotation Knee adduction Ankle dorsiflexor Lumbar extensor Lumbar bending Lumbar internal rotator 47.8 47.1 66.3 52.0 93.2 73.9 79.3 70.0 60.5 51.2 25.2 30.5 20.0 36.0 4.5 15.8 16.0 19.6 28.4 34.2 PC5 PC6 97.2 97.8 97.1 97.3 92.9 96.5 97.0 98.7 99.6 98.2 99.7 98.8 99.6 99.7 99.3 99.5 99.0 99.4 99.5 99.5 2.0 1.5 2.9 1.4 9.9 2.1 2.5 2.7 2.5 13.2 12.6 6.7 5.1 1.2 6.2 2.9 7.9 8.4 10.9 7.1 4.9 4.0 3.4 0.6 2.4 1.0 1.5 1.7 2.7 Total 4.1 3.1 1.8 2.1 0.7 1.6 99.0 98.3 98.8 98.7 99.6 98.3 99.2 99.0 99.0 99.7 Principal components retained after Weiss-Indurkhya independent feature selection are shown in bold italic text. Those subsequently retained after Sequential Feature Selection are shown with additional bold italic underline. Rows: Biomechanical variables input into the feature selection pipeline: F, muscle forces; h, joint angles; and M, joint moments. Columns: Variance explained by individual principal components up to the 6th principal component for each variable. PC abbreviates the term ‘‘principal component’’, and the numeric suffix is the number of that principal component, e.g. read PC3 as ‘‘third principle component’’. between control and ACLR groups at signiﬁcance level a ¼ 0:001. For each main feature, we qualitatively interpreted the inﬂuence of that feature (e.g., hKNEEFLEX PC1) on the original variable waveform (e.g., hKNEEFLEX), and described between-group differences, by: (1) plotting the waveforms of the original pooled data (ACLR and controls combined) corresponding to the upper and lower quartiles of the principal component scores (Figs. 1, 2, and 3, top row); (2) plotting the shape of the eigenvector (i.e, the waveform of the principal component coefﬁcients), and its squared correlation with the waveforms of the original data (i.e. variance explained by the feature, which naturally corresponds better with the upper quartile of the original pooled BIOMEDICAL ENGINEERING SOCIETY data) (Figs. 1, 2, and 3, bottom row); and (3) ﬁnally, we identiﬁed whether the ACLR group was ‘‘more like’’ the upper or the lower quartile based on the magnitude and sign of the mean principal component score compared to controls (Table 2). As this is a lengthy and exhaustive procedure, we provide a detailed step-by-step narrative description of the above process for each main feature as Supplementary Material, with the only summary of ﬁndings reported here (Table 3). All three principal components of muscle forces, FHAMS PC1 (read as ‘‘the ﬁrst principal component of the hamstrings force’’), FRF PC1 and FSOL PC1, differed between ACLR and controls at signiﬁcance level a ¼ 0:001 (Table 2). They were interpreted to upscale Hop-Landing Biomechanics After ACL-Reconstruction 335 TABLE 2. Group means and standard deviations of the principal component scores for 10 main features that best distinguish between ACLR and control groups during the landing phase of a single-leg forward hop. Feature FHAMS PC1 FRF PC1 FSOL PC1 hHIPROT PC2 hKNEEFLEX PC1 hKNEEFLEX PC3 hKNEEROT PC3 MKNEEROT PC3 MKNEEADD PC1 MLUMBARROT PC3 ACLR Control P g 0.62 (2.58) 2 1.63 (4.45) 2 1.94 (12.21) 2 1.70 (22.37) 2 11.32 (83.29) 2 2.09 (9.82) 2 0.99 (11.36) 2 0.11 (1.48) 5.52 (17.32) 0.27 (2.61) 2 1.25 (1.4) 3.31 (5.44) 3.79 (11.69) 3.8 (21.19) 27.08 (67.92) 3.4 (9.93) 2.2 (9.04) 0.22 (1.65) 2 12.06 (20.25) 2 0.59 (2.47) < 0.001 < 0.001 < 0.001 0.013 < 0.001 < 0.001 0.003 0.031 < 0.001 < 0.001 2 2 2 2 2 2 2 0.830 1.027 0.475 0.250 0.488 0.556 0.299 0.216 0.956 0.334 Principal component scores presented as mean (standard deviation). P-values calculated using t-test between ACLR and controls at an a priori significance level of a ¼ 0:001 as the 46 features input into Sequential Feature Selection were already significant at t 2:0 (approx. a ¼ 0:046Þ. Significant P-values are presented in bold italic. Effect sizes were calculated using Hedges g, with strong effects (jg j 0:8) shown in bold. FHAMS PC1 FRF PC1 2.5 Force (BW) Force (BW) Q1 (25th %tile) Q3 (75th %tile) 1 0.5 FSOL PC1 8 3 6 Force (BW) 1.5 2 1.5 1 4 2 0.5 0 0 100 0 % landing phase 0.14 0.6 0.08 0.4 PC coefficient Explained variance 0 50 % landing phase 0.6 0.1 0.4 0.2 0.05 0.2 100 0 0 100 50 % landing phase 50 100 % landing phase 0.8 PC coefficient 0.1 0.04 0 0.12 1 0.15 Explained variance PC coefficient 0.8 0.06 100 % landing phase 1 0.12 50 PC coefficient 50 Explained variance 0 1 0.1 0.8 0.08 0.6 0.06 0.4 0.04 0 50 Explained variance 0 0.2 100 % landing phase FIGURE 1. Main features representing muscle forces: FHAMS PC1, FRF PC1, and FSOL PC1. Top row: waveforms of the pooled original data (FHAMS, FRF and FSOL respectively), representing the upper (solid blue) and lower (dashed red) quartiles of the principal component scores for each respective feature. Shaded regions represent 1 standard deviation about the respective waveforms. Bottom row: for each feature, waveforms of the principal component coefficients (PC coefficient, solid black) and the squared correlation with the waveforms of the original data (Explained variance, dashed black). Landing phase: from foot strike (0%) to peak knee flexion angle (100%). their respective waveforms (i.e., increase the waveform amplitude), through middle of the landing phase, acting predominantly near their respective waveform peaks (Fig. 1; Table 3). Thus, these features inﬂuenced greater peak hamstrings force, but lower peak rectus femoris and soleus forces in the ACLR group. Two of the four principal components of joint angles, hKNEEFLEX PC1 and hKNEEFLEX PC3, differed between groups at signiﬁcance level a ¼ 0:001 (Table 2). hKNEEFLEX PC1 tended to downscale, i.e., reduce the amplitude, of the knee ﬂexion angle waveform throughout landing, while hKNEEFLEX PC3 further reBIOMEDICAL ENGINEERING SOCIETY SRITHARAN et al. θHIPROT PC2 20 θKNEEFLEX PC1 80 θKNEEFLEX PC3 80 15 0 40 20 -5 40 20 0 0 50 0.12 0.1 0.2 0.05 0.15 0.1 0 0.05 -0.05 -0.1 0 0 100 50 0.8 0.08 0.7 0.06 0.6 0.5 0.02 0 0.4 100 50 % landing phase 50 100 0 % landing phase 0.9 0.04 -20 -40 0 1 0.1 PC coefficient PC coefficient Explained variance 0.25 Explained variance 0.3 0.2 0.15 100 % landing phase 0.3 0.4 0.2 0.3 0.1 0.2 0 0.1 -0.1 0 % landing phase 50 100 % landing phase 0 100 50 0.25 0.2 PC coefficient 0 Explained variance 100 PC coefficient 50 % landing phase Explained variance 0 -10 -30 Q1 (25th %tile) Q3 (75th %tile) -10 PC coefficient Angle (deg) 5 0 60 Angle (deg) Angle (deg) Angle (deg) 60 10 θKNEEROT PC3 10 0.2 0.1 0.15 0 0.1 -0.1 0.05 -0.2 0 % landing phase 50 Explained variance 336 0 100 % landing phase FIGURE 2. Main features representing joint angles: hHIPROT PC2, hKNEEFLEX PC1, hKNEEFLEX PC3 and hKNEEROT PC3. Top row: waveforms of the pooled original data (hHIPROT, hKNEEFLEX, hKNEEFLEX and hKNEEROT respectively), representing the upper (solid blue) and lower (dashed red) quartiles of the principal component scores for each respective feature. Shaded regions represent 1 standard deviation about the respective waveforms. Bottom row: for each feature, waveforms of the principal component coefficients (PC coefficient, solid black) and the squared correlation with the waveforms of the original data (Explained variance, dashed black). Landing phase: from foot strike (0%) to peak knee flexion angle (100%). 2 1 0 -1 -2 Q1 (25th %tile) Q3 (75th %tile) 0 -5 -10 -15 100 % landing phase 0 0.05 -0.1 50 % landing phase -1 -2 100 0 0 100 0.2 1 0.1 0.6 0.4 0.05 PC coefficient Explained variance 0 0 50 % landing phase 50 100 % landing phase 0.8 PC coefficient 0.1 0 50 0.15 Explained variance PC coefficient 0.1 -0.2 0 % landing phase 0.15 0.2 1 -3 0 0.2 0 100 0.4 0.15 PC coefficient 50 Explained variance 0 MLUMBARROT PC3 2 0.3 0.1 0.05 0.2 0 0.1 -0.05 -0.1 0 50 Explained variance 3 Moment (%BWHT) Moment (%BWHT) MKNEEADD PC1 5 Moment (%BWHT) MKNEEROT PC3 4 0 100 % landing phase FIGURE 3. Main features representing joint moments: MKNEEROT PC3, MKNEEADD PC1, and MLUMBARROT PC3. Top row: waveforms of the pooled original data (MKNEEROT, MKNEEADD and MLUMBARROT respectively), representing the upper (solid blue) and lower (dashed red) quartiles of the principal component scores for each respective feature. Shaded regions represent 1 standard deviation about the respective waveforms. Bottom row: for each feature, waveforms of the principal component coefficients (PC coefficient, solid black) and the squared correlation with the waveforms of the original data (Explained variance, dashed black). Landing phase: from foot strike (0%) to peak knee flexion angle (100%). BIOMEDICAL ENGINEERING SOCIETY Hop-Landing Biomechanics After ACL-Reconstruction 337 TABLE 3. Qualitative interpretation of the effect of each principal component in the main feature set, and the corresponding differences between ACLR and control groups. Feature ACLR group :Nearest quartile Qualitative interpretation:The principal component… FHAMS PC1 …upscales the hamstrings force throughout middle of the landing phase, predominantly near the peak Upper FRF PC1 …upscales the rectus femoris force throughout middle of the landing phase, predominantly near the peak Lower FSOL PC1 Lower hKNEEROT PC3 …upscales the soleus force throughout middle of the landing phase, predominantly near the peak …increases hip internal rotation angle at foot strike, i.e. hip has greater range of motion through landing …downscales the waveform towards zero, reducing knee flexion angle, particularly after foot strike, i.e. favours straighter knee throughout landing …reduces instantaneous knee flexion angle at foot strike, i.e. at the instant of footstrike, tends to land with straighter knee initially …downscales the amplitude of oscillatory components knee rotation angle waveform MKNEEROT PC3 … applies a small modulation to the magnitude and timing of first peak of knee rotation moment waveform Lower MKNEEADD PC1 …downscales the knee adduction moment towards zero throughout the second half of the landing phase, predominantly near the peak …downscales the amplitude and frequency of oscillatory components of lumbar rotator moment waveform Upper hHIPROT PC2 hKNEEFLEX PC1 hKNEEFLEX PC3 MLUMBARROT PC3 Lower Lower Effect of principal componentà:The ACLR group tends to have… …greater peak hamstrings force, greater hamstrings force throughout the middle of landing …diminished peak rectus femoris force, smaller rectus femoris force throughout the middle of landing …diminished peak soleus force, smaller soleus force throughout the middle of landing …less range of motion throughout the landing phase …a straighter knee throughout landing phase, predominantly after foot strike Lower …a straighter knee at the instant of foot strike Lower …greater oscillations in knee rotation angle waveform, a more internally-rotated knee around mid-phase …more pronounced oscillations in knee rotation moment waveform, out of phase relative to controls …lower peak knee abduction moment, lower knee abduction moment throughout the middle of landing …less pronounced oscillations in lumbar rotator moment waveform, fewer peaks Upper * Qualitative interpretation: statement describing the meaning of the principal component with respect to its associated variable, i.e. description of the what the principal component does to the overall waveform of that variable. This is determined by analysing the shapes and magnitudes of waveforms of the upper and lower quartiles of the pooled data for that principal component, and comparing them against the waveforms of the principal component coefficient and the percentage of variance explained (Figs. 1, 2, and 3). ACLR group: indicates whether the mean principal component score for the ACLR group is nearer the upper or lower quartile of principal component scores for the pooled data, based on the principal component scores for each group (Table 2. à Effect of principal component: a statement describing how the principal component impacts the overall waveform for that variable in the ACLR group compared to the control group, based on: (1) the qualitative interpretation of the principal component; and (2) the quartile of the principal component scores that is nearest the average score for the ACLR group. duced the knee ﬂexion angle near foot strike (Fig. 2; Table 3). These features inﬂuenced a ‘‘straighter’’ a knee landing strategy in the ACLR group. Two of the three principal components of joint moments, MKNEEADD PC1 and MLUMBARROT PC3, differed signiﬁcantly between ACLR and controls at signiﬁcance level a ¼ 0:001 (Table 2). MKNEEADD PC1 tended to downscale the knee adduction moment waveform particularly near the peaks in the second half of the landing phase (Fig. 3; Table 3). The ACLR tended to land with less knee abduction moment compared to controls. MLUMBARROT PC3 tended to downscale both the amplitude and frequency of the lumbar rotator moment waveform, with the ACLR group having reduced range and frequency of lumbar rotator moments (Fig. 3; Table 3). Eight associated features were found that correlated moderately or strongly with the main features at signiﬁcance level a ¼ 0:05, and were also able to distinguish between ACLR and control groups at signiﬁcance level t 2:0 (approx. a ¼ 0:046Þ (Table 4), with six able to distinguish between groups at signiﬁcance level a ¼ 0:001. DISCUSSION Using automated pattern recognition methods, we found altered biomechanics in individuals 12-24 months after ACLR during the landing phase of a submaximal single-leg forward hop. We systematically identiﬁed and described 10 main features that distinBIOMEDICAL ENGINEERING SOCIETY 338 SRITHARAN et al. TABLE 4. Associated features for each main feature in the final set. Mean principal component scores Main feature Associated feature FHAMS PC1 FRF PC1 FSOL PC1 hHIPROT PC2 hKNEEFLEX PC1 None MKNEEFLEX PC1 MANKLEDF PC1 None FVAS PC1 MHIPROT PC2 MKNEEADD PC2 None None None hKNEEROT PC1 MHIPROT PC1 MLUMBARBEND PC3 hKNEEFLEX PC3 hKNEEROT PC3 MKNEEROT PC3 MKNEEADD PC1 MLUMBARROT PC3 ACLR Control P 0.638 2 0.862 7.67 3.09 2 15.23 2 5.96 < 0.001 < 0.001 1.084 0.347 2 0.536 2 0.524 0.577 2 2.52 0.55 2 1.24 5.45 2 0.83 1.93 < 0.001 0.002 < 0.001 2 0.712 0.311 2 0.347 2 0.618 2 0.611 0.510 2 7.57 2 2.33 0.49 19.66 4.98 2 1.21 < 0.001 < 0.001 0.002 2 0.484 2 0.989 0.304 q g Only associated features with moderate, 0:5 jqj<0:7, or strong. jqj 0:7, correlation with their main feature, and which were able to discriminate between ACLR and control groups at significance level t 2:0 (approx. a ¼ 0:046Þ are shown. Associated features with strong correlation are presented in bold italic with bold italic underline. All correlations are significant at a ¼ 0:05. P-values indicating significant between-group differences in principal component scores at level a ¼ 0:001 are presented in bold italic. Effect sizes for principal component scores were calculated using Hedges g, with strong, jg j 0:8, effects shown in bold. guished between the landing biomechanics of ACLR and control groups—speciﬁcally, 3 principal components of muscle forces, 4 of joint angles and 3 of joint moments, and 8 additional associated features—which advance and reinforce current understanding of altered biomechanics after ACLR. Furthermore, the ability to analyse full temporal waveforms rather than just discrete points, and also to embody a large suite of variables within a small set of main features, are two major advantages of using pattern recognition methods over traditional multivariate analyses. Our hypothesis was supported, as two principal components of knee ﬂexion angle were included as main features, and knee ﬂexor moment was included as an associated feature of rectus femoris force. Our pattern recognition method systematically detected large between-group diﬀerences in landing biomechanics, as evidenced by a number of high principal components (PC1, and some PC2) of variables in our main and associated feature sets, with the PC1 features accounting for between 52.0% and 93.3% of the variance in their respective variables. In particular, the ACLR group demonstrated considerably altered knee-spanning muscle forces and sagittalplane knee-joint biomechanics. Similar to reported ﬁndings for single-legged9,25,28,45 and double-legged20 landing tasks, individuals with ACLR landed with a more extended knee (hKNEEFLEX PC1) and reduced knee extensor moments (MKNEEFLEX PC1 via main feature FRF PC1). This latter ﬁnding manifested as diminished quadriceps forces in the ACLR group (FRF PC1, and FVAS PC1 via main feature hKNEEFLEX PC1). Together with greater hamstrings forces (FHAMS PC1) BIOMEDICAL ENGINEERING SOCIETY in the ACLR group, this indicated elevated hamstrings-quadriceps co-contraction throughout landing, a ﬁnding frequently reported in electromyographic studies in individuals with ACLR.42 Furthermore, these results could imply lower patellofemoral-joint loading in the ACLR group, which is associated with risk of future osteoarthritis.39,40 Our results are in agreement with those commonly reported for biomechanical alterations after ACLR surgery12,15,25,27,45 and our method could identify them with an exceptionally high degree of certainty (at the 99% conﬁdence level) providing conﬁdence in the validity of our approach. However, our ﬁnding of reduced ankle plantar ﬂexor function (FSOL PC1, and its associated feature MANKLEDF PC1) differs from previously reported results for landing activities, which found greater hip extension and ankle plantar ﬂexor moments in the ACLR limb relative to the uninjured contralateral limb9,28 during such tasks, typically described as a redistribution of torque away from the compromised knee.38 In our present study, reduced ankle plantar ﬂexor function with reduced knee (and hip; not selected as a main or associated feature; see Supplementary Material) extensor moments indicate a global reduction in kinetic demand across the ACLR limb. This could occur as a result of the straighter, stiffer landing strategy adopted by individuals with ACLR, which we have shown provides the same centre-ofmass modulation with less muscular effort, but at the expense of greater axial knee-joint loading.38 Our results may be explained by the reduced demands of our constrained sub-maximal single-leg forward hops, Hop-Landing Biomechanics After ACL-Reconstruction compared to the maximum-effort hops of Gokeler et al.,9 for example. We identiﬁed very subtle diﬀerences in the temporal waveforms of the biomechanical variables, and could say with at least 95% certainty that these were not simply due to measurement noise. This was evidenced by the inclusion of low principal components (PC3, and some PC2) in our main and associated feature sets, with PC3 features accounting for only 1.2% to 10.9% of the variance in those respective variables (Table 1). These small between-group differences could not be discerned by comparing group mean waveforms of the original data alone (Supplementary Material). Our results suggest that these subtle aberrations in nonsagittal-plane angles and moments may, in fact, be salient features of ACLR biomechanics 12-24 months post-surgery, that may not be easily detectable in a clinical setting. Speciﬁcally, these subtle biomechanical aberrations were indicated by the inclusion of low principal components of: (1) the knee (hKNEEFLEX PC3, hKNEEROT PC3 and MKNEEROT PC3), each of which accounted for less than 3% of the variance in their respective variables; (2) the hip (hHIPROT PC2), accounting for 8.9% of the variance of hip rotation angle; and (3) the back (MLUMBARROT PC3, MLUMBARBEND PC3), each of which accounted for less than 11% of the variance in their respective variables (Table 1). Both hKNEEFLEX PC3 and hHIPROT PC2 tended to perturb their respective joint angles at footstrike, while the others modulated oscillations in the temporal waveforms of their respective variables (Figs. 1, 2, and 3; Table 3), suggesting the ACLR group landed with a greater level of frontal- and transverse-plane unsteadiness or instability. Allowing individuals after ACLR to return to sport without speciﬁcally identifying and addressing these subtle instabilities may cause individuals to retain them as they increase the intensity and volume of their physical activity22, elevating risk of re-injury and future osteoarthritis. Currently, detecting these small aberrations reliably and robustly is challenging in a clinical setting, however, future technological advancements may enable such analyses to be undertaken routinely. Overall, non-sagittal-plane variables, speciﬁcally those associated with hip rotation, knee rotation and knee adduction angles and moments, were strongly represented in the main and associated feature sets (Tables 3 and 4). Most notably, our results add new evidence supporting the importance of abnormal/deﬁcient coordination of hip rotation in biomechanics of ACLR individuals.17,20,25 In the ACLR group, we found diminished internal hip rotator moments (MHIPROT PC1 and MHIPROT PC2) (Table 4), as well as a tendency for reduced range of hip rotation angle during landing (hHIPROT PC2). Holistically, these 339 ﬁndings suggest that reduced hip rotator muscle function in the ACLR group is associated with a more extended knee (hKNEEFLEX PC1), less internal rotation (hKNEEROT PC1), and diminished internal knee abduction moments (MKNEEADD PC1) (Tables 3 and 4). Thus, our results reinforce the role of dynamic coupling in hop-landing biomechanics,38 and the need to consider the entire kinetic chain in the assessment and rehabilitation of individuals after ACLR. Furthermore, deﬁciencies in hip external rotator strength can predict single-leg hop performance in ACLR individuals 8 months post-operatively,17 and, together with deﬁcient hip abductor muscle function, is a risk factor for lower-limb injuries during sports.19 Although hip external rotator muscles, such as gluteus medius, were not selected as either main or associated features, their deﬁcient action could be inferred from MHIPROT PC1, MHIPROT PC2 and hHIPROT PC2, and conﬁrmed by examining the actual group mean waveforms for gluteus medius (Supplementary Material). Additionally, it is possible that many individuals in our present ACLR cohort may have been at some risk of re-injury at the time of testing, as deﬁcient hip rotation kinetics strongly predicted ACL re-injury after returning to sport.30 Our present study is not without limitations. Firstly, PCA allows for examination of bimodal data (trial vs time, or variable vs time), whereas motion data is trimodal (trial vs variable vs time).11 A multimodal analysis method, such as Parallel Factor Analysis (PARAFAC), could potentially simultaneously identify the components describing individual or group differences in motion patterns, determine the instants when components are inﬂuential, and examine relationships between variables.11 However, the PCA calculation is a convex problem with unique solutions, while PARAFAC solves a non-convex problem that must, during its execution, examine locally-optimal solutions. Thus, overall PARAFAC is computationally more expensive than PCA, but not necessarily more accurate. Secondly, SFS is a suboptimal method for feature selection. It may be possible to ﬁnd a ‘‘better’’ set of discriminating features. However, feature selection problems may not be solved optimally in polynomial time37; therefore, only suboptimal methods such as SFS are feasible in practice.14 This implies that SFS, with our selection threshold of 10 features, could retain a few ‘‘weak’’ features while discarding some ‘‘stronger’’ ones. Yet, our use of t-tests at approximately 95% signiﬁcance level as a preliminary reduction method guarantees that all remaining features are statisticallyrelevant. Thirdly, the present set of results is valid only for distinguishing between groups using data from singleBIOMEDICAL ENGINEERING SOCIETY 340 SRITHARAN et al. leg hop landings. We chose to examine single-leg hop landings as they are an important task used the clinical assessment of ACLR individuals for return-to-sport. Nevertheless, our methodology is not task-speciﬁc. Inputting biomechanical data from other tasks, such as running or squatting, may reveal contrasting sets of discriminating features, which may be used to assess individuals’ performances in those tasks. Future studies may pool data from many tasks to reveal common discriminating features. Finally, our calculation of muscles forces did not consider subject-speciﬁc muscle activation patterns. We used CMC41 in OpenSim to calculate muscle activations, which can be constrained by inputting normalised electromyographic signals for one or more muscles if desired. However, electromyographic data were not collected in this study as it would have exacerbated the long testing time (>3 h) and participant fatigue. Nevertheless, our use of CMC, which combines static optimisation, forward dynamics and feedback control to calculate muscle activations, improves on previous estimates using static optimisation alone.39,40 In conclusion, we found altered landing biomechanics 12–24 months after ACLR using an automated pattern recognition approach. Our ﬁndings reinforce the importance of non-sagittal-plane biomechanical variables, as well as subtle aberrations, in clinical assessments after ACLR. Automated methods for classifying individuals with ACLR may, in future, have application in machine learning-based software tools for improving clinical decision-making around the time of return-to-sport. FUNDING Open Access funding enabled and organized by CAUL and its Member Institutions. Funding was provided by National Health & Medical Research Council (Dora Lush Postgraduate Scholarship and R.D. Wright Biomedical #1053521). SUPPLEMENTARY INFORMATION The online version contains supplementary material available at https://doi.org/10.1007/s10439-022-029214. ACKNOWLEDGMENTS L.P was a recipient of a National Health and Medical Research Council (NHMRC) Dora Lush Postgraduate Scholarship. A.L.B. is a recipient of an BIOMEDICAL ENGINEERING SOCIETY NHMRC Career Development Fellowship (R.D. Wright Biomedical, #1053521). CONFLICT OF INTEREST All authors declare no conﬂict of interest. No beneﬁts in any form have been or will be received from a commercial party related directly or indirectly to the subject of this manuscript. 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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https://openalex.org/W2052956178	https://journals.plos.org/plosntds/article/file?id=10.1371/journal.pntd.0001923&type=printable	English	null	Cathepsin B & L Are Not Required for Ebola Virus Replication	PLoS neglected tropical diseases	2,012	cc-by	8,873	Abstract Ebola virus (EBOV), family Filoviridae, emerged in 1976 on the African continent. Since then it caused several outbreaks of viral hemorrhagic fever in humans with case fatality rates up to 90% and remains a serious Public Health concern and biothreat pathogen. The most pathogenic and best-studied species is Zaire ebolavirus (ZEBOV). EBOV encodes one viral surface glycoprotein (GP), which is essential for replication, a determinant of pathogenicity and an important immunogen. GP mediates viral entry through interaction with cellular surface molecules, which results in the uptake of virus particles via macropinocytosis. Later in this pathway endosomal acidification activates the cysteine proteases Cathepsin B and L (CatB, CatL), which have been shown to cleave ZEBOV-GP leading to subsequent exposure of the putative receptor-binding and fusion domain and productive infection. We studied the effect of CatB and CatL on in vitro and in vivo replication of EBOV. Similar to previous findings, our results show an effect of CatB, but not CatL, on ZEBOV entry into cultured cells. Interestingly, cell entry by other EBOV species (Bundibugyo, Coˆte d’Ivoire, Reston and Sudan ebolavirus) was independent of CatB or CatL as was EBOV replication in general. To investigate whether CatB and CatL have a role in vivo during infection, we utilized the mouse model for ZEBOV. Wild-type (control), catB2/2 and catL2/2 mice were equally susceptible to lethal challenge with mouse-adapted ZEBOV with no difference in virus replication and time to death. In conclusion, our results show that CatB and CatL activity is not required for EBOV replication. Furthermore, EBOV glycoprotein cleavage seems to be mediated by an array of proteases making targeted therapeutic approaches difficult. Editor: Thomas Geisbert, University of Texas Medical Branch, United States of America Editor: Thomas Geisbert, University of Texas Medical Branch, United States of America Received July 13, 2012; Accepted October 12, 2012; Published December 6, 2012 This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Funding: This research was supported in part by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH) and the Public Health Agency of Canada (PHAC). A.M. was in part funded by the Natural Science and Engineering Council of Canada (NSERC). Introduction attachment factors seem to facilitate EBOV entry [3], so far no specific cell surface receptor molecule has been identified. Recently, it was shown that the cholesterol transporter Nie- mann-Pick C1 (NPC1) is required for ZEBOV infection [4,5]. NPC1 is a ubiquitously expressed endosomal membrane protein involved in the fusion and fission of endosomes and lysosomes [6], and its deficiency has been shown to impact on HIV-1 particle release [7]. Its presence in the endosome fits the current EBOV entry model based on macropinocytosis, a cellular pathway proposed to be the main uptake mechanism of EBOV particles into cells [8–10]. Previously, Chandran et al. identified the cysteine proteases cathepsin B (CatB) and cathepsin L (CatL), which are also present in endosomes, as important factors for ZEBOV entry [11]. According to the current model, cleavage of the ZEBOV glycoprotein (GP) by CatB is necessary for exposure of the core receptor-binding domain and fusion machinery, otherwise buried in the GP structure, to initiate fusion of the viral and the endosomal membrane [12–14]. Subsequently, the viral genome along with the replication complex is released into the cytoplasm where replication and virus progeny production occur. Members of the family Filoviridae, Ebola virus (EBOV) and Marburg virus (MARV), are the causative agents of viral hemorrhagic fever in central Africa and a major public health threat in their endemic areas. Worldwide concern relates to the importation of infected individuals and the potential use of filoviruses as biothreat pathogens [1]. All current MARV strains belong to the Lake Victoria marburgvirus species, while Ebola virus (EBOV) strains are attributed to five different species: Zaire ebolavirus (ZEBOV), Sudan ebolavirus (SEBOV), Coˆte d’Ivoire ebolavirus (CIE- BOV), Reston ebolavirus (REBOV) and Bundibugyo ebolavirus (BEBOV) [1,2]. The species vary in their pathogenicity for humans with ZEBOV being most pathogenic (up to 90% case fatality rate), followed by SEBOV and BEBOV with about 50% and .25% case fatality rates, respectively. CIEBOV and REBOV cause lethal infections in nonhuman primates, but have not yet been associated with fatal human cases [1,2]. Although EBOV (mainly ZEBOV) and MARV have been extensively studied in vitro and in vivo, today there is neither a licensed vaccine nor treatment available. EBOV entry into target cells is still not fully understood and remains a focus of ongoing research. While a number of CatB and CatL are members of a family of 11 human cysteine proteases. Abstract 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 ex * E-mail: feldmannh@niaid.nih.gov Andrea Marzi1,2, Thomas Reinheckel3,4, Heinz Feldmann1,2,5* 1 Laboratory of Virology, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana, United States of America, 2 Special Pathogens Program, National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, Manitoba, Canada, 3 Institute of Molecula Medicine and Cell Research, Albert-Ludwigs-University, Freiburg, Germany, 4 BIOSS Centre for Biological Signaling Studies, Albert-Ludwigs-University, Freiburg, Germany 5 Department of Medical Microbiology, University of Manitoba, Winnipeg, Manitoba, Canada Cathepsin B & L Are Not Required for Ebola Virus Replication Andrea Marzi1,2, Thomas Reinheckel3,4, Heinz Feldmann1,2,5* PLOS Neglected Tropical Diseases \| www.plosntds.org Foci reduction experiments p The endosomal acidification inhibitor Bafilomycin A1 (BafA1) and the inhibitor CA074 (specific for CatB) were obtained from Sigma (St. Louis, MO); CatL-inhibitor V was purchased from Calbiochem (via EMD Chemicals Inc., Gibbstown, NJ). All inhibitors were dissolved in DMSO and stored at 220uC. Dilutions were prepared in plain DMEM (no supplements) prior to every experiment. For foci reduction experiments Vero E6 cells were seeded in 48-well plates the day before infection. Media was removed and cells were incubated for one hour with 50 ml inhibitor in the following concentrations as described previously [11]: BafA1 200, 100, 50, 20 nM; CA074 (CatB) 200, 100, 50, 20 mM; CatL-inhibitor V 20, 10, 5, 2 mM. Then 50 ml plain DMEM with 100 focus forming units (ffu) virus were added and kept for one hour at 37uC. After three washes with plain DMEM a 1:1 mixture of 2.4% carboxymethyl cellulose (CMC) and 26 Minimal Essential Medium (MEM) (Life Technologies, Carlsbad, CA) supplemented with 4% FBS, penicillin/streptomycin and L- glutamine was added containing the appropriate concentration of inhibitor (BafA1 100, 50, 25, 10 nM; CA074 (CatB) 100, 50, 25, 10 mM; CatL-inhibitor V 10, 5, 2.5, 1 mM). SARS infected cells were fixed and stained with crystal violet and plaques were counted three days after infection. Four days after infection EBOV inoculated cells were fixed with 10% neutral buffered formalin and removed from BSL4 following standard operating procedures. Subsequently, the cells were permeabilized and foci were stained with a rabbit anti-VP40 antibody (kindly provided by Y. Kawaoka, University of Wisconsin, Madison, WI) or a rabbit serum directed against REBOV-NP (kindly provided by A. Takada, Hokkaido University, Sapporo, Japan) followed by a FITC-labeled secondary antibody (Sigma, ST. Louis, MO). Foci were counted using a fluorescent microscope (Carl Zeiss Micro- imaging LLC, Thornwood, NY). Here we show that while CatB mediates ZEBOV entry in vitro, it is not important for entry and replication of any other EBOV species. We further show that in vivo replication of mouse-adapted ZEBOV (MA-ZEBOV) is independent of CatB and CatL, indicating that both of these cathepsins are not required for ZEBOV replication in vivo. Animal ethics statement Animals were handled in the Biosafety Level 4 (BSL4) containment space of the Integrated Research Facility (IRF) at the Rocky Mountain Laboratories (RML), Division of Intramural Research (DIR), National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH). Research was conducted in compliance with the guidelines of the NIAID/ RML Institutional Animal Care and Use Committee (IACUC). The facility, where this research was conducted, is fully accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care International (AAALAC) with approved Office of Laboratory Animal Welfare (OLAW) assurance (#A4149-01). Research was conducted under a protocol approved by the IACUC. All procedures were conducted by trained personnel under veterinary supervision, and all invasive clinical Cell culture and virus propagation Vero E6 cells and Mouse Embryonic Fibroblast (MEF) cell lines lacking cathepsin B, cathepsin L or both cathepsins were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Sigma, St. Louis, MO) supplemented with 10% FBS, penicillin/streptomycin and L-glutamine in a 37uC incubator, 5% CO2. VSV (serotype Indiana), SARS (strain Tor 2), BEBOV [2], CIEBOV (strain Tai Forest), REBOV (strain Pennsylvania), SEBOV (strain Boniface), ZEBOV (strain Mayinga) and the furin knockout and mouse- adapted variants of ZEBOV (ZEBOV-Fko and MA-ZEBOV, respectively) [28–30] were propagated in Vero E6 cells. The supernatants were cleared of cell debris by centrifugation at 1,5006g for 10 min, aliquoted and stored in liquid nitrogen. Viral titers were determined conducting conventional plaque assay or immunoplaque assay. All infectious work with EBOV was performed in the Biosafety Level 4 (BSL4) laboratories at the National Microbiology Laboratory (NML) of the Public Health Agency of Canada (PHAC) or the IRF, RML, DIR, NIAID, NIH. and exhibit nonspecific proteolytic activity within lysosomes [15,16]. CatB has been associated with TNF-a induced liver damage and seems to play a critical role for the development of pancreatitis [17,18]. Despite these facts, catB2/2 mice are phenotypically similar to wild-type control mice and fully immunocompetent [18]. CatL is important for epidermal homeo- stasis and the regulation of the hair cycle and as such catL2/2 mice are hairless [19]. Furthermore, CatL is involved in MHC II- mediated antigen presentation in epithelial cells of the thymus [20]. Consequently, catL2/2 mice have reduced numbers of CD4+ T helper cells, which are however fully functional. A double knockout mouse lacking CatB and CatL has been generated but is not viable long enough for experimental use [21,22]. To determine the importance of cathepsins in viral infections, various inhibitors of endosomal acidification, cathepsin or specifically CatB and CatL activity have been used in vitro. In these studies the role of cathepsins have been demonstrated to be important for the entry of reovirus, SARS-CoV, henipaviruses and ZEBOV [11,14,23–27]. Author Summary It is currently believed that Ebola virus (EBOV) enters cells via macropinocytosis following which, the cysteine prote- ases cathepsin B and L (CatB, CatL) cleave the viral glycoprotein (GP) allowing exposure of its core receptor- binding and fusion domain thus facilitating subsequent infection. We studied the effect of CatB and CatL on in vitro and in vivo EBOV replication. Our results demonstrate a reduction of Zaire ebolavirus (ZEBOV) entry upon selective inhibition of CatB, but not CatL in cell culture. Interestingly, all other EBOV species enter the cells efficiently when CatB and/or CatL activity is blocked. Moreover, when wild-type (control), catB2/2 and catL2/2 mice were infected with a lethal dose of mouse-adapted ZEBOV, all animals were equally susceptible to lethal challenge with no difference in virus replication and time to death. Therefore, we conclude that EBOV replication is dispensable of CatB and CatL, and proteolytic processing of GP can also be mediated by other endosomal proteases. Introduction Both proteases are highly abundant, broadly expressed December 2012 \| Volume 6 \| Issue 12 \| e1923 1 PLOS Neglected Tropical Diseases \| www.plosntds.org December 2012 \| Volume 6 \| Issue 12 \| e1923 Ebola Viruses Replicate Cathepsin B/L-Independent procedures were performed while animals were anesthetized. Endpoint criteria, as specified by the IACUC approved scoring parameters, were used to determine when animals should be humanely euthanized. Author Summary Ebola Viruses Replicate Cathepsin B/L-Independent Ebola Viruses Replicate Cathepsin B/L-Independent plain DMEM, cells in each well were covered with 1 ml DMEM (supplemented with 2% FBS) containing 50 nM BafA1, 50 mM CA074, 5 mM CatL-inhibitor V or no inhibitor. MEF cell lines were seeded in a 24-well plate the day before the experiment. For infection, 200 ml ZEBOVwt or MA-ZEBOV (MOI = 1) were added and incubated for one hour. Following three washes with plain DMEM, cells in each well were covered with 1 ml DMEM (supplemented with 2% FBS). At time points 0, 12, 24, 48, 72 and 96 hours post infection 200 ml supernatant were collected from all infected cells, and 200 ml DMEM with 2% FBS containing the appropriate concentration of inhibitor were added back into each well. Samples were stored at 280uC before titration on Vero E6 cells. plain DMEM, cells in each well were covered with 1 ml DMEM (supplemented with 2% FBS) containing 50 nM BafA1, 50 mM CA074, 5 mM CatL-inhibitor V or no inhibitor. MEF cell lines were seeded in a 24-well plate the day before the experiment. For infection, 200 ml ZEBOVwt or MA-ZEBOV (MOI = 1) were added and incubated for one hour. Following three washes with plain DMEM, cells in each well were covered with 1 ml DMEM (supplemented with 2% FBS). At time points 0, 12, 24, 48, 72 and 96 hours post infection 200 ml supernatant were collected from all infected cells, and 200 ml DMEM with 2% FBS containing the appropriate concentration of inhibitor were added back into each well. Samples were stored at 280uC before titration on Vero E6 cells. based on foci reduction using the same inhibitors. Cells were seeded and treated with various lysosome and cathepsin inhibitors for one hour, infected with ZEBOV wild-type (wt) for one hour, covered with carboxymethyl cellulose containing inhibitors and fixed after incubation for 4 days. The number of foci was determined by immunostaining. Only the inhibition of CatB resulted in significantly reduced virus entry, whereas CatL proteolytic activity did not appear to be obligatory for ZEBOV uptake into Vero E6 cells, although CatL is highly expressed in these cells [27]. SARS-CoV was used as a CatL-dependent control virus [26] to verify the activity of the CatL inhibitor (Fig. 1). Virus load determination Vero E6 cells were seeded in 96-well plates the day before titration. Liver and spleen samples were thawed, weighed, homogenized in 10-fold weight/volume of plain DMEM and serial 10-fold dilutions were prepared. Blood samples and supernatant collected from infected cells to determine viral growth for ZEBOVwt and MA-ZEBOV were thawed and serial 10-fold dilutions were prepared. Media was removed from cells and triplicates were inoculated with each dilution. After one hour DMEM supplemented with 2% FBS, penicillin/streptomycin and L-glutamine was added and incubated at 37uC. Cells were monitored for cytopathic effect (CPE) and 50% tissue culture infectious dose (TCID50) was calculated for each sample employ- ing the Reed and Muench method [31]. Humoral immune responses in mice For the detection of ZEBOV-GP specific antibodies in mouse sera, soluble ZEBOV-GP antigen (ZEBOV-GPDTM) was pro- duced and used in an enzyme-linked immunosorbent assay (ELISA) as described before [32,33]. For the detection of VSV- specific antibodies VSV was propagated in Vero E6 cells. VSV particles were harvested after 48 hrs, purified by centrifugation through a 20% sucrose cushion and treated with 0.05% Triton- X100 in PBS prior to storage at 280uC. The ELISA used VSV antigen in a 1:100 dilution. Sera from mice infected with MA- ZEBOV were inactivated by c-irradiation as per standard operating protocol. Statistical analyses One-way ANOVA was performed using Prism 5 (Graph Pad Software Inc.). Mouse infections Groups of C57BL/6 catB2/2, C57BL/6 catL2/2 or C57BL/6 (control) mice were infected intraperitoneally (i.p.) with 10 ffu MA- ZEBOV (1,000 LD50) or 16105 pfu VSVwt (serotype Indiana) and monitored daily for weight loss and signs of disease. On day 3 and 7 post infection 3 mice of each group were euthanized and blood, liver and spleen samples were collected and stored at 280uC for virus titration. Surviving animals were euthanized at day 28 (study endpoint) and final serum was collected to determine antibody titers. Ebola Viruses Replicate Cathepsin B/L-Independent In addition to ZEBOVwt, we also tested a furin cleavage site knockout ZEBOV (ZEBOV-Fko) [28,29] and the MA-ZEBOV in this assay to determine whether furin cleavage of the glycoprotein or mutations caused by the adaptation process of the virus to mice would influence the requirement for cathepsin cleavage. Both viruses entered Vero E6 cells in a CatB-dependent manner similar to ZEBOVwt and were not affected by the presence of the CatL inhibitor (Fig. 1). These results show that the uptake of all tested viruses occurred by utilizing the cellular endocytotis machinery as indicated by the inhibitory effect of Bafilomycin A1 (BafA1), an endosomal acidification inhibitor. Furthermore, proteolytic pro- cessing of the ZEBOV-GP by furin is not a prerequisite for cathepsin cleavage in the virus maturation or entry process. In addition, mutations throughout the viral genome acquired during the adaptation process of ZEBOV to the mouse have no influence on ZEBOV uptake. ZEBOV replication is largely independent on cathepsin B cleavage After confirmation that CatB has a role in ZEBOV entry we analyzed virus replication in the presence of cathepsin inhibitors. Vero E6 cells were treated with inhibitors (BafA1 100 nM; CatB 100 mM; CatL 10 mM) for one hour prior to ZEBOVwt infection (MOI = 1). After unbound virus was washed away the cells were covered with medium containing inhibitor (BafA1 50 nM; CatB 50 mM; CatL 5 mM), samples were taken at the indicated time points and stored at 280uC for virus titration. Virus titers were determined on Vero E6 cells using a 50% tissue culture infectious dose (TCID50) assay and calculated using the Reed and Muench formula (Fig. 3A) [31]. The results demonstrate that ZEBOV replicates to similar titers in the presence of CatB or CatL inhibitor (difference less than 1 log); the presence of both inhibitors reduced The role of CatB in cellular uptake differs among Ebola virus species Our data on ZEBOV only partially support previously published observations [11], but confirm more recent studies claiming that CatB, rather than CatL, plays a role in ZEBOV entry [34,35]. Therefore, we studied the role of cathepsin cleavage on virus entry for BEBOV, CIEBOV, REBOV and SEBOV using a representative strain for each EBOV species. Foci reduction assays were performed and evaluated as described above. Infection of all viruses was reduced in the presence of BafA1 (Fig. 2), confirming that endosomal acidification plays a critical role during the entry process of all these EBOVs. Interestingly, only the maximum concentration of the CatB inhibitor had an effect on BEBOV uptake into Vero E6 cells, whereas CIEBOV, REBOV and SEBOV produced foci independent of the presence of the inhibitor (Fig. 2). The entry of none of the EBOVs was affected by the CatL inhibitor. This data suggests that CatB is not equally important for the entry of other EBOVs and seems to have a selective effect on ZEBOV and a limited effect on BEBOV entry. Viral growth kinetics Vero E6 cells were seeded in a 24-well plate the day before the experiment. Pretreatment occurred with 200 ml of 100 nM BafA1, 100 mM CA074, 10 mM CatL-inhibitor V or no inhibitor for 1 hour. Thereafter, 200 ml ZEBOVwt (MOI = 1) were added and cells were incubated for another hour. Following three washes with December 2012 \| Volume 6 \| Issue 12 \| e1923 PLOS Neglected Tropical Diseases \| www.plosntds.org 2 The number of foci and plaques without inhibitor was set as 100%. A representative experiment performed in triplicates is shown. Error bars indicate the standard error of the mean. ZEBOVwt = Zaire ebolavirus, strain Mayinga; ZEBOV-Fko = Zaire ebolavirus furin cleavage site knockout mutant; MA-ZEBOV = mouse-adapted Zaire ebolavirus. doi:10.1371/journal.pntd.0001923.g001 observed at certain time points, one-way ANOVA did not find statistically significant p values (Fig. 3). ZEBOV replication by about 1 log. This indicates that the CatB inhibitory effect on entry is likely compensated for by other cellular proteases. However, BafA1 reduces virus growth by more than 2 logs, showing that endosomal acidification is important for efficient ZEBOV entry and replication (Fig. 1, 3A). In order to further confirm the data we performed ZEBOVwt and MA- ZEBOV infections in MEF cell lines deficient in the expression of CatB, CatL or both cathepsins. The cells were infected for one hour with ZEBOVwt or MA-ZEBOV (MOI = 1) and samples were taken at the indicated time points. Virus titers were determined on Vero E6 cells using a TCID50 assay and calculated using the Reed and Muench formula (Fig. 3B,C) [31]. ZEBOVwt and MA-ZEBOV replicated similarly well in the absence of CatB or CatL or both proteases. Although small differences in titer were ZEBOV replication by about 1 log. This indicates that the CatB inhibitory effect on entry is likely compensated for by other cellular proteases. However, BafA1 reduces virus growth by more than 2 logs, showing that endosomal acidification is important for efficient ZEBOV entry and replication (Fig. 1, 3A). In order to further confirm the data we performed ZEBOVwt and MA- ZEBOV infections in MEF cell lines deficient in the expression of CatB, CatL or both cathepsins. The cells were infected for one hour with ZEBOVwt or MA-ZEBOV (MOI = 1) and samples were taken at the indicated time points. Virus titers were determined on Vero E6 cells using a TCID50 assay and calculated using the Reed and Muench formula (Fig. 3B,C) [31]. ZEBOVwt and MA-ZEBOV replicated similarly well in the absence of CatB or CatL or both proteases. Although small differences in titer were CatB2/2 and catL2/2 mice succumb to ZEBOV infection Finally, the in vitro data were verified in vivo using the well- established lethal mouse model for ZEBOV [36]. ZEBOV entry in vitro is CatB-mediated The observation that entry of ZEBOV into Vero E6 cells is CatB- and CatL-dependent was initially described in 2005 by Chandran and colleagues [11]. For further analysis of ZEBOV uptake into Vero E6 cells we performed a different in vitro assay December 2012 \| Volume 6 \| Issue 12 \| e1923 PLOS Neglected Tropical Diseases \| www.plosntds.org 3 Figure 1. Zaire ebolavirus entry is CatB mediated. Vero E6 cells were treated prior to infection for one hour with inhibitors directed against th indicated cathepsin(s) or endosomal acidification (BafA1). Following infection the cells were washed and a carboxymethyl cellulose overlay wa added containing inhibitor (BafA1 100, 50, 25, 10 nM; CA-074 (CatB) 100, 50, 25, 10 mM; CatL inhibitor V 10, 5, 2.5, 1 mM). SARS-CoV infected cells wer Ebola Viruses Replicate Cathepsin B/L-Independen Ebola Viruses Replicate Cathepsin B/L-Independent Figure 1. Zaire ebolavirus entry is CatB mediated. Vero E6 cells were treated prior to infection for one hour with inhibitors directed against the indicated cathepsin(s) or endosomal acidification (BafA1). Following infection the cells were washed and a carboxymethyl cellulose overlay was added containing inhibitor (BafA1 100, 50, 25, 10 nM; CA-074 (CatB) 100, 50, 25, 10 mM; CatL inhibitor V 10, 5, 2.5, 1 mM). SARS-CoV infected cells were fixed on day 3, stained with crystal violet and plaques were counted. ZEBOV infected cells were fixed on day 4, foci were stained with an antibody against ZEBOV-VP40 and counted. The number of foci and plaques without inhibitor was set as 100%. A representative experiment performed in triplicates is shown. Error bars indicate the standard error of the mean. ZEBOVwt = Zaire ebolavirus, strain Mayinga; ZEBOV-Fko = Zaire ebolavirus furin cleavage site knockout mutant; MA-ZEBOV = mouse-adapted Zaire ebolavirus. doi:10.1371/journal.pntd.0001923.g001 Figure 1. Zaire ebolavirus entry is CatB mediated. Vero E6 cells were treated prior to infection for one hour with inhibitors directed against the indicated cathepsin(s) or endosomal acidification (BafA1). Following infection the cells were washed and a carboxymethyl cellulose overlay was added containing inhibitor (BafA1 100, 50, 25, 10 nM; CA-074 (CatB) 100, 50, 25, 10 mM; CatL inhibitor V 10, 5, 2.5, 1 mM). SARS-CoV infected cells were fixed on day 3, stained with crystal violet and plaques were counted. ZEBOV infected cells were fixed on day 4, foci were stained with an antibody against ZEBOV-VP40 and counted. The number of foci and plaques without inhibitor was set as 100%. A representative experiment performed in triplicates is shown. Error bars indicate the standard error of the mean. ZEBOVwt = Zaire ebolavirus, strain Mayinga; ZEBOV-Fko = Zaire ebolavirus furin cleavage site knockout mutant; MA-ZEBOV = mouse-adapted Zaire ebolavirus. doi:10.1371/journal.pntd.0001923.g001 Figure 1. Zaire ebolavirus entry is CatB mediated. Vero E6 cells were treated prior to infection for one hour with inhibitors directed against the indicated cathepsin(s) or endosomal acidification (BafA1). Following infection the cells were washed and a carboxymethyl cellulose overlay was added containing inhibitor (BafA1 100, 50, 25, 10 nM; CA-074 (CatB) 100, 50, 25, 10 mM; CatL inhibitor V 10, 5, 2.5, 1 mM). SARS-CoV infected cells were fixed on day 3, stained with crystal violet and plaques were counted. ZEBOV infected cells were fixed on day 4, foci were stained with an antibody against ZEBOV-VP40 and counted. Figure 2. Entry of other Ebola viruses is CatB-independent. Vero E6 cells were treated prior to infection for one hour with inhibitors directed against the indicated cathepsin(s) or endosomal acidification (BafA1). Following infection the cells were washed and a carboxymethyl cellulose overlay containing inhibitor (BafA1 100, 50, 25, 10 nM; CA-074 (CatB) 100, 50, 25, 10 mM; CatL inhibitor V 10, 5, 2.5, 1 mM) was added. After fixation on day 4, foci were stained with antibodies against VP40 (BEBOV = Bundibugyo ebolavirus, CIEBOV = Coˆte d’Ivoire ebolavirus, SEBOV = Sudan ebolavirus, strain Boniface) or NP (REBOV = Reston ebolavirus, strain Pennsylvania) and counted. The number of foci without inhibitor was set as 100%. A representative experiment performed in triplicates is shown. Error bars indicate the standard error of the mean. doi:10.1371/journal.pntd.0001923.g002 Figure 2. Entry of other Ebola viruses is CatB-independent. Vero E6 cells were treated prior to infection for one hour with inhibitors directed against the indicated cathepsin(s) or endosomal acidification (BafA1). Following infection the cells were washed and a carboxymethyl cellulose overlay containing inhibitor (BafA1 100, 50, 25, 10 nM; CA-074 (CatB) 100, 50, 25, 10 mM; CatL inhibitor V 10, 5, 2.5, 1 mM) was added. After fixation on day 4, foci were stained with antibodies against VP40 (BEBOV = Bundibugyo ebolavirus, CIEBOV = Coˆte d’Ivoire ebolavirus, SEBOV = Sudan ebolavirus, strain Boniface) or NP (REBOV = Reston ebolavirus, strain Pennsylvania) and counted. The number of foci without inhibitor was set as 100%. A representative experiment performed in triplicates is shown. Error bars indicate the standard error of the mean. doi:10.1371/journal.pntd.0001923.g002 Figure 2. Entry of other Ebola viruses is CatB-independent. Vero E6 cells were treated prior to infection for one hour with inhibitors directed against the indicated cathepsin(s) or endosomal acidification (BafA1). Following infection the cells were washed and a carboxymethyl cellulose overlay containing inhibitor (BafA1 100, 50, 25, 10 nM; CA-074 (CatB) 100, 50, 25, 10 mM; CatL inhibitor V 10, 5, 2.5, 1 mM) was added. After fixation on day 4, foci were stained with antibodies against VP40 (BEBOV = Bundibugyo ebolavirus, CIEBOV = Coˆte d’Ivoire ebolavirus, SEBOV = Sudan ebolavirus, strain Boniface) or NP (REBOV = Reston ebolavirus, strain Pennsylvania) and counted. The number of foci without inhibitor was set as 100%. A representative experiment performed in triplicates is shown. Error bars indicate the standard error of the mean. The catB2/2 and catL2/2 mice are well characterized [37] and therefore ideal to determine the importance of CatB and CatL for ZEBOV replication in vivo. MEF cell lines obtained from these mice have been used here and in previous studies to demonstrate that mouse cathepsins are functionally similar to human cathepsins and important for ZEBOV-GP-mediated entry [11,38]. Groups of catB2/2, catL2/2 and control mice were infected with 1,000 LD50 of MA-ZEBOV and monitored daily for signs of disease including December 2012 \| Volume 6 \| Issue 12 \| e1923 PLOS Neglected Tropical Diseases \| www.plosntds.org 4 Ebola Viruses Replicate Cathepsin B/L-Independent Figure 2. Entry of other Ebola viruses is CatB-independent. Vero E6 cells were treated prior to infection for one hour with inhibitors directed against the indicated cathepsin(s) or endosomal acidification (BafA1). Following infection the cells were washed and a carboxymethyl cellulose PLOS Neglected Tropical Diseases \| www.plosntds.org doi:10.1371/journal.pntd.0001923.g002 similar among all three mouse strains but higher in the blood of catB2/2 and catL2/2 mice compared with wt mice (Fig. 5B). This indicates that disease progression and outcome were similar in all the animals (Fig. 4, 5). In order to exclude the occurrence of mutations in the glycoprotein (GP) gene of MA-ZEBOV during in vivo replication, we determined the full GP sequence and found no mutations (data not shown). weight loss (Fig. 4A,B). On day 3 and 7 post-infection 3 mice in each group were euthanized and blood, liver and spleen samples were taken to determine the viral load (Fig. 5). All animals in the catL2/2 group succumbed to MA-ZEBOV infection between days 7 and 9, similar to most control mice. All but one of the 22 catB2/2 mice challenged with MA-ZEBOV succumbed to infection; the one surviving mouse showed signs of disease and recovered (Fig. 4A,B). As previously observed, the infection of control mice is not always uniformly lethal [33]. Here 3 out of 22 control mice developed disease but survived the challenge (Fig. 4A,B). There were no significant differences between viral titers in liver, spleen and blood samples taken on day 3 post MA- ZEBOV infection from the three different mouse strains (Fig. 5A). At day 7 post Ma-ZEBOV infection liver and spleen titers were To exclude that catB2/2 and catL2/2 mice are in general more susceptible to viral infections, groups of mice were infected with recombinant vesicular stomatitis virus (VSV), strain Indiana, and monitored daily for weight loss and signs of illness (Fig. 4C). VSV did not cause disease in any of the mice demonstrating that neither the catB2/2 nor the catL2/2 mice do possess an increased susceptibility to virus infection. In blood, liver or spleen samples December 2012 \| Volume 6 \| Issue 12 \| e1923 PLOS Neglected Tropical Diseases \| www.plosntds.org 5 Ebola Viruses Replicate Cathepsin B/L-Independent Figure 3. Zaire ebolavirus growth is CatB and CatL-independent. (A) Vero E6 cells were seeded the night before infection in a 24-well-p One hour prior to infection cells were incubated with inhibitors directed against the indicated cathepsin(s) or endosomal acidification (Baf ZEBOVwt was added at an MOI of 1 and incubated for one hour at 37uC. After three washes the cells were covered with 1 ml medium contai 50 nM BafA1, 50 mM CA-074, 5 mM CatL inhibitor V or no inhibitor and incubated for 4 days. (B) and (C) MEF cell lines were seeded the night be PLOS Neglected Tropical Diseases \| www.plosntds.org 6 December 2012 \| Volume 6 \| Issue 12 \| e Figure 3. Zaire ebolavirus growth is CatB and CatL-independent. (A) Vero E One hour prior to infection cells were incubated with inhibitors directed against ZEBOVwt was added at an MOI of 1 and incubated for one hour at 37uC. After thr 50 nM BafA1, 50 mM CA-074, 5 mM CatL inhibitor V or no inhibitor and incubated fo PLOS Neglected Tropical Diseases \| www.plosntds.org 6 Figure 3. Zaire ebolavirus growth is CatB and CatL-independent. (A) Vero E6 cells were seeded the night before infection in a 24-well-plate. One hour prior to infection cells were incubated with inhibitors directed against the indicated cathepsin(s) or endosomal acidification (Baf A1). ZEBOVwt was added at an MOI of 1 and incubated for one hour at 37uC. After three washes the cells were covered with 1 ml medium containing 50 nM BafA1, 50 mM CA-074, 5 mM CatL inhibitor V or no inhibitor and incubated for 4 days. (B) and (C) MEF cell lines were seeded the night before PLOS Neglected Tropical Diseases \| www.plosntds.org PLOS Neglected Tropical Diseases \| www.plosntds.org December 2012 \| Volume 6 \| Issue 12 \| e1923 6 Ebola Viruses Replicate Cathepsin B/L-Independent Ebola Viruses Replicate Cathepsin B/L-Independent infection in a 24-well-plate. Cells were infected for 1 hour with 0.2 ml ZEBOVwt (B) or MA-ZEBOV (C) at a MOI of 1. After three washes the cells were covered with 1 ml medium and incubated for 4 days. For all experiments, samples were collected at 0, 12, 24, 48, 72 and 96 hours post infection and infectious titers were determined. A representative experiment performed in triplicates is shown. Error bars indicate the standard error of the mean. doi:10.1371/journal.pntd.0001923.g003 infection in a 24-well-plate. Cells were infected for 1 hour with 0.2 ml ZEBOVwt (B) or MA-ZEBOV (C) at a MOI of 1. After three washes the cells were covered with 1 ml medium and incubated for 4 days. For all experiments, samples were collected at 0, 12, 24, 48, 72 and 96 hours post infection and infectious titers were determined. A representative experiment performed in triplicates is shown. Error bars indicate the standard error of the mean. doi:10.1371/journal.pntd.0001923.g003 taken on day 3 and 7 post VSV infection no viral RNA was detected (data not shown), indicating that catB2/2 and catL2/2 as well as control mice were able to efficiently clear the virus. ELISA performed with serum samples of these mice showed that all animals were infected as indicated by the detection of VSV- specific antibodies (Fig. 4D). This data demonstrates that there is no obvious difference between catB2/2, catL2/2 and control mice in susceptibility to viral infections and the development of immune responses. supports more recent studies showing that only CatB mediates ZEBOV entry into target cells [34,35]. In addition, we could demonstrate that post-translational furin cleavage of ZEBOV-GP into the fragments GP1 and GP2 is not a prerequisite for cathepsin processing. This result does not come as a big surprise considering earlier studies reporting that ZEBOV replication and pathogenic- ity were independent of furin cleavage [28,29]. Interestingly, all other EBOV species tested in our study seem to enter Vero E6 cells in a CatB- and CatL-independent manner suggesting that other endosomal proteases might functionally replace CatB in virus entry. This finding is in disagreement with recently published data showing that cell entry of VSV- and HIV- 1-based pseudotype particles expressing different EBOV-GPs is CatB-dependent [38,39]. Interestingly, one of these studies also showed that cell entry of infectious SEBOV (strain Gulu) was CatB- and CatL-independent as we could demonstrate here for a Discussion The present study demonstrates that CatB, but not CatL, mediates ZEBOV uptake into Vero E6 cells. This observation is only partially in line with the initial in vitro studies demonstrating both CatB and CatL dependent uptake of ZEBOV [11,14] and Figure 4. CatB2/2 and catL2/2 mice succumb to Ebola virus but not to VSV infection. Groups of mice were i.p. infected with 10 ffu MA- ZEBOV (1,000 LD50) or 16105 pfu VSV (serotype Indiana) and monitored daily for weight loss and other signs of illness. Survival (A) and weight curves (B) for MA-ZEBOV infection are shown. Body weights of VSV-infected mice are shown in (C). VSV antibodies were detected using ELISA to confirm infection (D). doi:10.1371/journal.pntd.0001923.g004 Figure 4. CatB2/2 and catL2/2 mice succumb to Ebola virus but not to VSV infection. Groups of mice were i.p. infected with 10 ffu MA- ZEBOV (1,000 LD50) or 16105 pfu VSV (serotype Indiana) and monitored daily for weight loss and other signs of illness. Survival (A) and weight curves (B) for MA-ZEBOV infection are shown. Body weights of VSV-infected mice are shown in (C). VSV antibodies were detected using ELISA to confirm infection (D). doi:10.1371/journal.pntd.0001923.g004 December 2012 \| Volume 6 \| Issue 12 \| e1923 December 2012 \| Volume 6 \| Issue 12 \| e1923 7 Ebola Viruses Replicate Cathepsin B/L-Independent different SEBOV strain (strain Boniface) [38]. The discrepancies d t d f th i l bli h d t as well as the mechanism of particle uptake. HIV-1 particles are l l h i l d th h i f t k i t Figure 5. Zaire ebolavirus replicates to similar titers in knockout and control mice. CatB2/2, catL2/2 or control mice (n = 3) were i.p. infected with 1,000 LD50 of MA-ZEBOV and euthanized at the indicated time point. Liver, spleen and blood samples were taken on day 3 (A) and day 7 (B) and viral titers were determined. A single 50% tissue culture infectious dose (TCID50) value is depicted for each mouse. Bars indicate the mean value. doi:10.1371/journal.pntd.0001923.g005 Ebola Viruses Replicate Cathepsin B/L-Independent Figure 5. Zaire ebolavirus replicates to similar titers in knockout and control mice. CatB2/2, catL2/2 or control mice (n = 3) were i.p. infected with 1,000 LD50 of MA-ZEBOV and euthanized at the indicated time point. Liver, spleen and blood samples were taken on day 3 (A) and day 7 (B) and viral titers were determined. References 12. Schornberg K, Matsuyama S, Kabsch K, Delos S, Bouton A, et al. (2006) Role of endosomal cathepsins in entry mediated by the Ebola virus glycoprotein. J Virol 80: 4174–4178. 1. Sanchez A, Geisbert TW, Feldmann H (2006) Filoviridae: Marburg and Ebola viruses Fields Virology: 1409–1448. 2. Towner JS, Sealy TK, Khristova ML, Albarino CG, Conlan S, et al. (2008) Newly discovered ebola virus associated with hemorrhagic fever outbreak in Uganda. PLoS Pathog 4: e1000212. 13. Kaletsky RL, Simmons G, Bates P (2007) Proteolysis of the Ebola virus glycoproteins enhances virus binding and infectivity. J Virol 81: 13378–13384. 3. Takada A (2012) Filovirus tropism: cellular molecules for viral entry. Frontiers in Microbiology 3/00034. 14. Sanchez A (2007) Analysis of filovirus entry into vero e6 cells, using inhibitors of endocytosis, endosomal acidification, structural integrity, and cathepsin (B and L) activity. J Infect Dis 196 Suppl 2: S251–258. 4. Carette JE, Raaben M, Wong AC, Herbert AS, Obernosterer G, et al. (2011) Ebola virus entry requires the cholesterol transporter Niemann-Pick C1. Nature 477: 340–343. 15. Rossi A, Deveraux Q, Turk B, Sali A (2004) Comprehensive search for cysteine cathepsins in the human genome. Bio Chem 385: 363–372. 5. Cote M, Misasi J, Ren T, Bruchez A, Lee K, et al. (2011) Small molecule inhibitors reveal Niemann-Pick C1 is essential for Ebola virus infection. Nature 477: 344–348. 16. Barrett AJ (1992) Cellular proteolysis. An overview. Annals of the New York Academy of Sciences 674: 1–15. 17. Guicciardi ME, Deussing J, Miyoshi H, Bronk SF, Svingen PA, et al. (2000) Cathepsin B contributes to TNF-alpha-mediated hepatocyte apoptosis by promoting mitochondrial release of cytochrome c. J Clin Invest 106: 1127–1137. 6. Goldman SD, Krise JP (2010) Niemann-Pick C1 functions independently of Niemann-Pick C2 in the initial stage of retrograde transport of membrane- impermeable lysosomal cargo. J Biol Chem 285: 4983–4994. 18. Halangk W, Lerch MM, Brandt-Nedelev B, Roth W, Ruthenbuerger M, et al. (2000) Role of cathepsin B in intracellular trypsinogen activation and the onset of acute pancreatitis. J Clin Invest 106: 773–781. 7. Tang Y, Leao IC, Coleman EM, Broughton RS, Hildreth JE (2009) Deficiency of niemann-pick type C-1 protein impairs release of human immunodeficiency virus type 1 and results in Gag accumulation in late endosomal/lysosomal compartments. J Virol 83: 7982–7995. 19. Roth W, Deussing J, Botchkarev VA, Pauly-Evers M, Saftig P, et al. Discussion In contrast, all knockout and C57BL/6 mice infected with MA-ZEBOV succumbed to infection with no difference in disease progression and time to death (Fig. 4A) or viral loads in liver, spleen and blood (Fig. 5) demonstrating that MA-ZEBOV replication in vivo is CatB- and CatL-independent. gp160 with CD4 and CCR5 or CXCR4 [40]. VSV particles are short and bullet-shaped and cell uptake occurs via the endocytic pathway [41]. In the infectious ZEBOV context the interactions of GP with VP24 and VP40 (missing in pseudotype particles) may further influence the cellular uptake mechanism [14], altogether suggesting that HIV-1- and VSV-based pseudotype particle entry could be different from those of filovirus particles, which are extremely long and filamentous in shape and mainly utilize macropinocytosis for particle uptake [8–10]. In addition, the CIEBOV-GP used to produce VSV-based pseudotype particles in one study lacked the mucin-like domain, which could have had impact on the GP structure and thus might have affected cleavage and entry [38]. In our view this highlights the need for confirmation of data obtained from pseudotype particle systems by live EBOV infections or at least by the use of EBOV-like particles. Finally, cell type and origin may also influence CatB- and CatL-mediated cleavage as studies were performed in different cell lines. ZEBOV into Vero E6 cells was CatB-dependent but CatL- independent (Fig. 1). Furthermore, both EBOVs replicated to high titers in MEF cell lines independent of CatB and/or CatL (Fig. 3B,C). Therefore, we used the mouse disease model to investigate the effect of these cathepsins on ZEBOV replication in vivo. C57BL/6 mice (genetic background), CatB or CatL knockout mice (catB2/2 or catL2/2) did not show an increased susceptibility to viral infection in general as determined here with VSV (Fig. 4C,D). In contrast, all knockout and C57BL/6 mice infected with MA-ZEBOV succumbed to infection with no difference in disease progression and time to death (Fig. 4A) or viral loads in liver, spleen and blood (Fig. 5) demonstrating that MA-ZEBOV replication in vivo is CatB- and CatL-independent. In conclusion, our studies indicate that CatB and CatL are not absolutely required for EBOV replication. For yet unknown reasons, CatB seems to play a more considerable role in ZEBOV uptake than it does for any other EBOV species. EBOV seems to have evolved to use a broader spectrum of endosomal proteases to ensure GP cleavage and thus facilitates successful infection of target cells. Acknowledgments We thank Hideki Ebihara (DIR, NIAID, NIH) for critical discussion throughout this study and Darryl Falzarano (DIR, NIAID, NIH) for reviewing the manuscript. The authors thank Julie Callison (DIR, NIAID, NIH) for excellent technical assistance and are grateful to Laura Tally (RMVB, NIAID, NIH) for assistance with the knockout mice. We thank Kathleen Meuchel, Jayne Faris, and Sandy Skorupa (RMVB, NIAID, NIH) for assistance with animal care in BSL4. Opinions, interpretations, conclusions, and recommendations are those of the authors and are not necessarily endorsed by NIH or PHAC. Author Contributions g p p y g Previous reports have shown that EBOV-GPs were also processed by cathepsins in MEFs indicating that mouse CatB and CatL are functionally active [11,38]. Here we have demonstrated that, similarly to ZEBOVwt, cell entry by MA- g p p y g Previous reports have shown that EBOV-GPs were also processed by cathepsins in MEFs indicating that mouse CatB and CatL are functionally active [11,38]. Here we have demonstrated that, similarly to ZEBOVwt, cell entry by MA- Conceived and designed the experiments: AM HF. Performed the experiments: AM HF. Analyzed the data: AM HF. Contributed reagents/materials/analysis tools: TR. Wrote the paper: AM TR HF. Conceived and designed the experiments: AM HF. Performed the experiments: AM HF. Analyzed the data: AM HF. Contributed reagents/materials/analysis tools: TR. Wrote the paper: AM TR HF. Discussion Therefore, therapeutic approaches targeting single proteases are unlikely to be beneficial to combat EBOV infections. For SARS-CoV entry, which is reported to be highly CatL- dependent, it has been shown that expression of the cellular transmembrane protease serine 2 (TMPRSS2) can overcome the block in SARS-CoV infection and replication caused by CatL inhibitors [42–45]. Moreover, signaling of toll-like receptor 9 (TLR9) has initially been associated with CatB, CatL and CatK activities [46,47]. However, studies using bone marrow derived macrophages and dendritic cells derived from cathepsin knockout mice did not identify a single cathepsin as an essential factor for TLR9 signaling [48,49] and rather point towards a role of other endolysosomal proteases, such as asparagine endopeptidase (AEP) [50] for activation. Thus, it seems reasonable to speculate that in the absence of CatB and CatL, such as in corresponding knockout mice, other endosomal proteases will mediate EBOV-GP cleavage enabling cathepsin-independent EBOV entry into target cells. Discussion A single 50% tissue culture infectious dose (TCID50) value is depicted for each mouse. Bars indicate the mean value. doi:10.1371/journal.pntd.0001923.g005 diff S BO i ( i B if ) h di i ll h h i f i l k i l Figure 5. Zaire ebolavirus replicates to similar titers in knockout and control mice. CatB2/2, catL2/2 or control mice (n = 3) were i.p. infected with 1,000 LD50 of MA-ZEBOV and euthanized at the indicated time point. Liver, spleen and blood samples were taken on day 3 (A) and day 7 (B) and viral titers were determined. A single 50% tissue culture infectious dose (TCID50) value is depicted for each mouse. Bars indicate the mean value. doi:10.1371/journal.pntd.0001923.g005 Figure 5. Zaire ebolavirus replicates to similar titers in knockout and control mice. CatB2/2, catL2/2 or control mice (n = 3) were i.p. infected with 1,000 LD50 of MA-ZEBOV and euthanized at the indicated time point. Liver, spleen and blood samples were taken on day 3 (A) and day 7 (B) and viral titers were determined. A single 50% tissue culture infectious dose (TCID50) value is depicted for each mouse. Bars indicate the mean value. doi:10.1371/journal.pntd.0001923.g005 as well as the mechanism of particle uptake. HIV-1 particles are largely spherical and the mechanism of uptake is receptor mediated through the interaction of its surface glycoprotein different SEBOV strain (strain Boniface) [38]. The discrepancies among our data and some of the previously published reports might be explained by the differences in size and shape of particles different SEBOV strain (strain Boniface) [38]. The discrepancies among our data and some of the previously published reports might be explained by the differences in size and shape of particles December 2012 \| Volume 6 \| Issue 12 \| e1923 December 2012 \| Volume 6 \| Issue 12 \| e1923 PLOS Neglected Tropical Diseases \| www.plosntds.org 8 Ebola Viruses Replicate Cathepsin B/L-Independent ZEBOV into Vero E6 cells was CatB-dependent but CatL- independent (Fig. 1). Furthermore, both EBOVs replicated to high titers in MEF cell lines independent of CatB and/or CatL (Fig. 3B,C). Therefore, we used the mouse disease model to investigate the effect of these cathepsins on ZEBOV replication in vivo. C57BL/6 mice (genetic background), CatB or CatL knockout mice (catB2/2 or catL2/2) did not show an increased susceptibility to viral infection in general as determined here with VSV (Fig. 4C,D). Ebola Viruses Replicate Cathepsin B/L-Independent 23. Ebert DH, Deussing J, Peters C, Dermody TS (2002) Cathepsin L and cathepsin B mediate reovirus disassembly in murine fibroblast cells. J Biol Chem 277: 24609–24617. 37. Reiser J, Adair B, Reinheckel T (2010) Specialized roles for cysteine cathepsins in health and disease. J Clin Invest 120: 3421–3431. 38. Misasi J, Chandran K, Yang JY, Considine B, Filone CM, et al. (2012) Filoviruses require endosomal cysteine proteases for entry but exhibit distinct protease preferences. J Virol 86: 3284–3292. 24. Chandran K, Nibert ML (2003) Animal cell invasion by a large nonenveloped virus: reovirus delivers the goods. Trends Micro 11: 374–382. g 25. Pager CT, Dutch RE (2005) Cathepsin L is involved in proteolytic processing of the Hendra virus fusion protein. J Virol 79: 12714–12720. p p 39. Gnirss K, Kuhl A, Karsten C, Glowacka I, Bertram S, et al. (2012) Cathepsins B and L activate Ebola but not Marburg virus glycoproteins for efficient entry into cell lines and macrophages independent of TMPRSS2 expression. Virology 424: 3–10. the Hendra virus fusion protein. J Virol 79: 12714–12720. 26. Simmons G, Gosalia DN, Rennekamp AJ, Reeves JD, Diamond SL, et al. (2005) Inhibitors of cathepsin L prevent severe acute respiratory syndrome coronavirus entry. Proc Natl Acad Sci USA 102: 11876–11881. 40. Wilen CB, Tilton JC, Doms RW (2012) Molecular Mechanisms of HIV entry. Adv Exp Med Biol 726: 223–242. y 27. Diederich S, Sauerhering L, Weis M, Altmeppen H, Schaschke N, et al. (2012) Activation of the Nipah virus fusion protein in MDCK cells is mediated by cathepsin B within the endosomal-recycling compartment. J Virol. 41. Albertini AA, Baquero E, Ferlin A, Gaudin Y (2012) Molecular and cellular aspects of rhabdovirus entry. Viruses 4: 117–139. 42. Bertram S, Glowacka I, Muller MA, Lavender H, Gnirss K, et al. (2011) Cleavage and activation of the severe acute respiratory syndrome coronavirus spike protein by human airway trypsin-like protease. J Virol 85: 13363–13372. 28. Neumann G, Feldmann H, Watanabe S, Lukashevich I, Kawaoka Y (2002) Reverse genetics demonstrates that proteolytic processing of the Ebola virus glycoprotein is not essential for replication in cell culture. Journal of virology 76: 406–410. 43. Glowacka I, Bertram S, Muller MA, Allen P, Soilleux E, et al. (2011) Evidence that TMPRSS2 activates the severe acute respiratory syndrome coronavirus spike protein for membrane fusion and reduces viral control by the humoral immune response. J Virol 85: 4122–4134. 29. Ebola Viruses Replicate Cathepsin B/L-Independent Neumann G, Geisbert TW, Ebihara H, Geisbert JB, Daddario-DiCaprio KM, et al. (2007) Proteolytic processing of the Ebola virus glycoprotein is not critical for Ebola virus replication in nonhuman primates. Journal of virology 81: 2995– 2998. 44. Matsuyama S, Nagata N, Shirato K, Kawase M, Takeda M, et al. (2010) Efficient activation of the severe acute respiratory syndrome coronavirus spike protein by the transmembrane protease TMPRSS2. J Virol 84: 12658–12664. 30. Bray M (2001) The role of the Type I interferon response in the resistance of mice to filovirus infection. J Gen Virol 82: 1365–1373. J 31. Reed LJ, Muench H (1938) A simple method of estimating fifty percent endpoints. The AMerican Journal of Hygiene 27: 493–497. p y p J 45. Shulla A, Heald-Sargent T, Subramanya G, Zhao J, Perlman S, et al. (2011) A transmembrane serine protease is linked to the severe acute respiratory syndrome coronavirus receptor and activates virus entry. J Virol 85: 873–882. 32. Nakayama E, Yokoyama A, Miyamoto H, Igarashi M, Kishida N, et al. (2010) Enzyme-linked immunosorbent assay for the detection of filovirus species- specific antibodies. Clin Vaccine Immunol. y p y J 46. Asagiri M, Hirai T, Kunigami T, Kamano S, Gober HJ, et al. (2008) Cathepsin K-dependent toll-like receptor 9 signaling revealed in experimental arthritis. Science 319: 624–627. p 33. Marzi A, Ebihara H, Callison J, Groseth A, Williams KJ, et al. (2011) Vesicular stomatitis virus-based Ebola vaccines with improved cross-protective efficacy. J Infect Dis 204 Suppl 3: S1066–1074. 47. Matsumoto F, Saitoh S, Fukui R, Kobayashi T, Tanimura N, et al. (2008) Cathepsins are required for Toll-like receptor 9 responses. Biochem Biophys Res Commun 367: 693–699. 34. Wong AC, Sandesara RG, Mulherkar N, Whelan SP, Chandran K (2010) A forward genetic strategy reveals destabilizing mutations in the Ebolavirus glycoprotein that alter its protease dependence during cell entry. J Virol 84: 163– 175. 48. Ewald SE, Lee BL, Lau L, Wickliffe KE, Shi GP, et al. (2008) The ectodomain of Toll-like receptor 9 is cleaved to generate a functional receptor. Nature 456: 658–662. 35. Martinez O, Johnson J, Manicassamy B, Rong L, Olinger GG, et al. (2010) Zaire Ebola virus entry into human dendritic cells is insensitive to cathepsin L inhibition. Cellular microbiology 12: 148–157. 49. Park B, Brinkmann MM, Spooner E, Lee CC, Kim YM, et al. References (2000) Cathepsin L deficiency as molecular defect of furless: hyperproliferation of keratinocytes and pertubation of hair follicle cycling. The FASEB journal : official publication of the Federation of American Societies for Experimental Biology 14: 2075–2086. 8. Saeed MF, Kolokoltsov AA, Albrecht T, Davey RA (2010) Cellular entry of ebola virus involves uptake by a macropinocytosis-like mechanism and subsequent trafficking through early and late endosomes. PLoS Pathog 6: e1001110. 9. Nanbo A, Imai M, Watanabe S, Noda T, Takahashi K, et al. (2010) Ebolavirus is internalized into host cells via macropinocytosis in a viral glycoprotein- dependent manner. PLoS Pathog 6: e1001121. 20. Nakagawa T, Roth W, Wong P, Nelson A, Farr A, et al. (1998) Cathepsin L: critical role in Ii degradation and CD4 T cell selection in the thymus. Science 280: 450–453. p g 10. Aleksandrowicz P, Marzi A, Biedenkopf N, Beimforde N, Becker S, et al. (2011) Ebola virus enters host cells by macropinocytosis and clathrin-mediated endocytosis. J Infect Dis 204 Suppl 3: S957–967. 21. Sevenich L, Pennacchio LA, Peters C, Reinheckel T (2006) Human cathepsin L rescues the neurodegeneration and lethality in cathepsin B/L double-deficient mice. Biol Chem 387: 885–891. 22. Felbor U, Kessler B, Mothes W, Goebel HH, Ploegh HL, et al. (2002) Neuronal loss and brain atrophy in mice lacking cathepsins B and L. Proc Natl Acad Sci USA 99: 7883–7888. 11. Chandran K, Sullivan NJ, Felbor U, Whelan SP, Cunningham JM (2005) Endosomal proteolysis of the Ebola virus glycoprotein is necessary for infection. Science 308: 1643–1645. December 2012 \| Volume 6 \| Issue 12 \| e1923 December 2012 \| Volume 6 \| Issue 12 \| e1923 PLOS Neglected Tropical Diseases \| www.plosntds.org 9 PLOS Neglected Tropical Diseases \| www.plosntds.org Ebola Viruses Replicate Cathepsin B/L-Independent Ebola Viruses Replicate Cathepsin B/L-Independent Ebola Viruses Replicate Cathepsin B/L-Independent (2008) Proteolytic cleavage in an endolysosomal compartment is required for activation of Toll-like receptor 9. Nature immunology 9: 1407–1414. gy 36. Bray M, Davis K, Geisbert T, Schmaljohn C, Huggins J (1999) A mouse model for evaluation of prophylaxis and therapy of Ebola hemorrhagic fever. J Infect Dis 179 Suppl 1: S248–258. p gy 50. Ewald SE, Engel A, Lee J, Wang M, Bogyo M, et al. (2011) Nucleic acid recognition by Toll-like receptors is coupled to stepwise processing by cathepsins and asparagine endopeptidase. J Exp Med 208: 643–651. 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https://openalex.org/W2974770100	https://inserm.hal.science/inserm-02479829v2/document	English	null	Long-term immunity against yellow fever in children vaccinated during infancy: a longitudinal cohort study	Lancet. Infectious diseases/The Lancet. Infectious diseases	2,019	cc-by	11,193	To cite this version: Cristina Domingo, Juliane Fraissinet, Patrick O Ansah, Corey Kelly, Niranjan Bhat, et al.. Long-term immunity against yellow fever in children vaccinated during infancy: a longitudinal cohort study. The Lancet Infectious Diseases, 2019, 19 (12), pp.1363-1370. 10.1016/S1473-3099(19)30323-8. inserm- 02479829v2 Long-term immunity against yellow fever in children vaccinated during infancy: a longitudinal cohort study Cristina Domingo, Juliane Fraissinet, Patrick O Ansah, Corey Kelly, Niranjan Bhat, Samba Sow, José E Mejía HAL Id: inserm-02479829 https://inserm.hal.science/inserm-02479829v2 Submitted on 14 Feb 2020 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Articles Long-term immunity against yellow fever in children vaccinated during infancy: a longitudinal cohort study Cristina Domingo, Juliane Fraissinet, Patrick O Ansah, Corey Kelly, Niranjan Bhat, Samba O Sow, José E Mejía Cristina Domingo, Juliane Fraissinet, Patrick O Ansah, Corey Kelly, Niranjan Bhat, Samba O Sow, José E Mejí Lancet Infect Dis 2019; 19: 1363–70 Copyright Copyright © 2019 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license. Summary y Background A single dose of vaccine against yellow fever is routinely administered to infants aged 9–12 months under the Expanded Programme on Immunization, but the long-term outcome of vaccination in this age group is unknown. We aimed to evaluate the long-term persistence of neutralising antibodies to yellow fever virus following routine vaccination in infancy. Lancet Infect Dis 2019; 19: 1363–70 Lancet Infect Dis 2019; 19: 1363–70 Published Online September 19, 2019 https://doi.org/10.1016/ S1473-3099(19)30323-8 See Comment page 1275 Robert Koch Institute, Highly Pathogenic Viruses (ZBS 1), Centre for Biological Threats and Special Pathogens, WHO Collaborating Centre for Emerging Infections and Biological Threats, Berlin, Germany (C Domingo, J Fraissinet); Navrongo Health Research Centre and Research Laboratory, Navrongo, Ghana (P O Ansah); PATH, Seattle, WA, USA (C Kelly, N Bhat); National Institute of Research on Public Health, Bamako, Mali (S A Sow); and Centre de Physiopathologie Toulouse-Purpan (CNRS, INSERM, Université Paul Sabatier), Centre Hospitalier Universitaire Purpan, Toulouse, France (J E Mejía) Correspondence to: Dr Cristina Domingo, Robert Koch Institute, Highly Pathogenic Viruses (ZBS 1), Centre for Biological Threats and Special Pathogens, WHO Collaborating Centre for Emerging Infections and Biological Threats, Berlin 13353, Germany domingo-carrascoc@rki.de Methods We did a longitudinal cohort study, using a microneutralisation assay to measure protective antibodies against yellow fever in Malian and Ghanaian children vaccinated around age 9 months and followed up for 4·5 years (Mali), or 2·3 and 6·0 years (Ghana). Healthy children with available day-0 sera, a complete follow-up history, and no record of yellow fever revaccination were included; children seropositive for yellow fever at baseline were excluded. We standardised antibody concentrations with reference to the yellow fever WHO International Standard. Findings We included 587 Malian and 436 Ghanaian children vaccinated between June 5, 2009, and Dec 26, 2012. In the Malian group, 296 (50·4%, 95% CI 46·4–54·5) were seropositive (antibody concentration ≥0·5 IU/mL) 4·5 years after vaccination. Among the Ghanaian children, 121 (27·8%, 23·5–32·0) were seropositive after 2·3 years. These results show a large decrease from the proportions of seropositive infants 28 days after vaccination, 96·7% in Mali and 72·7% in Ghana, reported by a previous study of both study populations. The number of seropositive children increased to 188 (43·1%, 95% CI 38·5–47·8) in the Ghanaian group 6·0 years after vaccination, but this result might be confounded by unrecorded revaccination or natural infection with wild yellow fever virus during a 2011–12 outbreak in northern Ghana. Interpretation Rapid waning of immunity during the early years after vaccination of 9-month-old infants argues for a revision of the single-dose recommendation for this target population in endemic countries. The short duration of immunity in many vaccinees suggests that booster vaccination is necessary to meet the 80% population immunity threshold for prevention of yellow fever outbreaks. Funding Wellcome Trust. www.thelancet.com/infection Vol 19 December 2019 Evidence before this study ﬂaviviruses can be discounted, have reported a 30–40% decrease in vaccine-induced immunity 5–10 years after yellow fever vaccination. Infants, and children in general, exhibit a higher vaccine failure and lower immune responses after vaccination than adults, but previous studies on the outcome of yellow fever vaccination have focused mainly on adults or older children. Studies covering vaccinated infants have been limited to determining seroconversion 1–3 months after vaccination. y WHO has recommended a single lifetime vaccination against yellow fever since 2013, following published evidence of long-lasting immunogenicity of the YF-17D vaccine. The requirement for a booster dose every 10 years was accordingly removed from the International Health Regulations in 2016. A shortcoming of this policy change is the scarcity of information on the eﬀective duration of protective immunity elicited in vaccinated infants, even though this age group constitute the main vaccination target in yellow fever-endemic countries. We consulted reference documents prepared by WHO and by the Centers for Disease Control and Prevention on yellow fever vaccination and the primary sources on the duration of yellow fever vaccine immunity surveyed from four review articles from 2013–16, including one meta-analysis and one systematic review. We also searched PubMed on May 18, 2018, with the expressions “yellow fever immunity” and “children” or “infants”, “yellow fever immunity” and “persistence”, and “yellow fever vaccine”, and did a reverse search on the Web of Science database, for articles citing three highly relevant research reports. We did not apply any language or date restrictions. Although the yellow fever vaccine can elicit lifelong immunity, studies in healthy adults have observed a time-dependent decrease in the proportion of seropositive individuals to 71–82% 10 years or more after vaccination, down from the proportion of more than 90% usually detected during the ﬁrst year. Studies in non-endemic settings, where interference from circulating Methods Time elapsed since vaccination Age at sample collection Median, years Range, days Median (IQR), days Range, months Median (IQR), months Mean (SD), years Ghana, year 2·3 2·3 733–874 823 (815–830) 36–38 36 (36–36) 3·0 (0·01) Ghana, year 6·0 6·0 2048–2331 2180 (2142–2219) 77–85 80 (79–82) 6·7 (0·1) Mali, year 4·5 4·5 1444–1793 1626 (1514–1688) 56–68 62 (59–64) 5·2 (0·3) Table 1: Schedule of sample collections after yellow fever vaccination Implications of all the available evidence p Our ﬁndings argue for the one-dose-for-life guidelines to be reconsidered for individuals who receive yellow fever vaccination as infants. The long-term decline of humoral immunity suggests that a single dose of the vaccine, administered at 9 months of age, might not achieve a population immunity protective against yellow fever epidemics. See Online for appendix www.thelancet.com/infection Vol 19 December 2019 Added value of this study We show a large decline in humoral immunity to yellow fever after 2–6 years in each group of children relative to earlier observations 4 weeks after vaccination. Proportions of seropositive children approximately halved, leaving large proportions of the study populations with a negative serostatus. These results address an important knowledge gap and are informative on the evolution of conferred immunity, in two diﬀerent African settings endemic for yellow fever. Introduction immunity in areas of yellow fever trans mission, which makes outbreaks more frequent.3 It is estimated that 393·7–472·9 million people will require vaccination4 to achieve the population immunity recommended by WHO for countries at risk.1 Yellow fever is a persistent public health problem and a growing concern in 34 African countries and 13 countries in the Americas.1 The re-emergence of yellow fever has led to the largest outbreak in Africa of the past 20 years: the Angola outbreak of December, 2015, connected with the 2016 outbreak in the neighbouring Democratic Republic of the Congo. During the outbreak that started in Brazil in 2016, the virus spread into areas that were not previously considered at high risk, including the densely populated periphery of the large cities of São Paulo, Rio de Janeiro, and Salvador de Bahia. The vaccines against yellow fever are safe and eﬃcacious and consist of live attenuated virus that is usually administered by subcutaneous injection. WHO guidelines advocate a single dose of vaccine for life-long protective immunity against yellow fever.5 In endemic countries, the vaccine is routinely given to infants at 9–12 months of age as part of the Expanded Programme on Immunization. The merits of one-dose vaccination at such early ages have not yet been supported by evidence that shows vaccine-elicited immunity to yellow fever persisting for many years in the absence of booster doses. Several studies have shown a decrease in seropositivity and antibody titres in vaccines over time, with 71–82% of adults seropositive 10 years or more after vaccination.6 Studies on the loss of immunity in The evolving epidemiology of the disease and the expansion of at-risk areas have been associated with prolonged periods of increased rainfall and temperatures and with environmental perturbations arising from human activity (eg, deforestation, population movements, and changes in land use).2 The resurgence of yellow fever, however, has been attributed largely to lapses of continuous vaccination coverage and the waning of population 1363 Articles Articles See Online for appendix Study design and participants Percentage of subjects and 95% CI 20 30 40 50 60 70 80 0·022 <0·0001 Positive Mali, 4·5 years Ghana, 2·3 years Ghana, 6·0 years Positive or borderline Mali, 4·5 years Ghana, 2·3 years Ghana, 6·0 years Prevalence p value 50·4 27·8 43·1 69·7 39·4 51·1 46·4–54·5 23·5–32·0 38·5–47·8 66·0–73·4 34·9–44·0 46·5–55·8 N 587 436 436 587 436 436 n % 95% CI 296 121 188 409 172 223 <0·0001 <0·0001 <0·0001 <0·0001 re 1: Forest plots of the prevalence of seropositive children Figure 1: Forest plots of the prevalence of seropositive children Seropositive participants at the antibody concentration threshold of 0·5 IU/mL or more are shown at the top of the ﬁgure, and seropositive and borderline study participants are presented at the bottom as a merged category including all participants with a measurable titre. p values test the diﬀerences of proportions between groups. Figure 1: Forest plots of the prevalence of seropositive children Seropositive participants at the antibody concentration threshold of 0·5 IU/mL or more are shown at the top of the ﬁgure, and seropositive and borderline study participants are presented at the bottom as a merged category including all participants with a measurable titre. Study design and participants We did a longitudinal cohort study. We studied two groups of healthy children: one from Ghana and one from Mali. Both groups are subsets of the study populations in trials of the meningococcal group A conjugate vaccine, MenAfriVac, administered to infants in association with other local Expanded Programme on Immunization vaccines, namely the PsATT-004 (phase 2, ISRCTN82484612) and Pers-004 (phase 4, ISRCTN10763234) studies in Ghana and the PsATT-007 (phase 3, PACTR201110000328305) and Pers-007 (phase 4, ISRCTN37623829) studies in Mali (appendix p 2). adults done in non-endemic settings, where a boost of vaccine-induced immunity by later natural infections is unlikely, have reported that up to 30%–40% of individuals serorevert 5–10 years after vaccination.6 Children, however, showed lower sero conversion rates and titres than healthy adults and might lose immunity faster.7 ( ) The children were concomitantly vaccinated under the Expanded Programme on Immunization schedule against yellow fever and measles at 8–12 months of age. Ghanaian children received the YF-17DD yellow fever vaccine strain (Bio-Manguinhos-Fiocruz, Rio de Janeiro, Brazil), and Malian children were vaccinated with the YF-17D-213 strain (Chumakov Institute of Poliomyelitis and Viral Encephalitides of the Russian Academy of Sciences, Moscow, Russia). The children provided serum samples either around 2·3 and 6·0 years (PsATT-004 and Pers-004, Ghana) or 4·5 years (Pers-007, Mali) after vaccination (table 1). Only healthy children with available Longitudinal studies of vaccine immunogenicity are essential to determine the risk of vaccination failure in individuals and to verify whether the single-dose policy guarantees community-level immunity above the 80% protective threshold against outbreaks. We evaluated the long-term persistence of neutralising antibodies to yellow fever virus following routine vaccination in infants aged around 9 months. 1364 Articles Figure 1: Forest plots of the prevalence of seropositive children Seropositive participants at the antibody concentration threshold of 0·5 IU/mL or more are shown at the top of the ﬁgure, and seropositive and borderline study participants are presented at the bottom as a merged category including all participants with a measurable titre. p values test the diﬀerences of proportions between groups. Study design and participants p values test the diﬀerences of proportions b t Participants Neutralising antibodies (IU/mL) n (%, 95% CI) Percentage of seropositive participants Geometric mean (95% CI) Median (IQR) Seropositive participants (antibody concentration ≥0·5 IU/ml) Mali, year 4·5, n=587 Total 296 (50·4%, 46·4–54·5) 100·0% 1·120 (1·040–1·210) 0·915 (0·702–1·440) Low tier 242 (41·2%) 81·8% 0·863 (0·826–0·902) 0·882 (0·610–1·210) High tier 54 (9·2%) 18·2% 3·530 (3·100–4·070) 2·890 (2·420–4·640) Ghana, year 2·3, N=436 Total 121 (27·8%, 23·5–32·0) 100·0% 1·380 (1·170–1·660) 1·020 (0·721–2·030) Low tier 90 (20·6%) 74·4% 0·854 (0·792–0·922) 0·765 (0·610–1·210) High tier 31 (7·1%) 25·6% 5·560 (4·110–7·790) 4·080 (2·890–8·160) Ghana, year 6·0, n=436 Total 188 (43·1%, 38·5–47·8) 100·0% 1·790 (1·550–2·080) 1·440 (0·854–2·870) Low tier 110 (25·2%) 58·5% 0·915 (0·856–0·978) 0·911 (0·647–1·210) High tier 78 (17·9%) 41·5% 4·610 (3·810–5·670) 2·890 (2·350–7·800) Borderline participants (measurable antibody concentration <0·5 IU/ml) Mali, year 4·5 113 (19·3%, 16·1–22·4) NA 0·335 (0·318–0·353) 0·361 (0·271–0·453) Ghana, year 2·3 51 (11·7%, 8·7–14·7) NA 0·359 (0·337–0·382) 0·381 (0·312–0·430) Ghana, year 6·0 35 (8·0%, 5·5–10·6) NA 0·334 (0·309–0·361) 0·361 (0·302–0·383) Broadly seropositive participants (all participants with a measurable antibody concentration) Mali, year 4·5 409 (69·7%, 66·0–73·4) NA 0·801 (0·742–0·865) 0·721 (0·457–1·220) Ghana, year 2·3 172 (39·4%, 34·9–44·0) NA 0·926 (0·796–1·090) 0·721 (0·455–1·400) Ghana, year 6·0 223 (51·1%, 46·5–55·8) NA 1·380 (1·190–1·600) 1·170 (0·640–2·360 ) The low and high tiers of seropositive participants are diﬀerentiated at the 1·8 IU/ml or more threshold. NA=not applicable. Table 2: Antibody concentration strata for participants with measurable neutralising antibodies day-0 sera, a complete follow-up history, and no record of yellow fever revaccination were included; children who were seropositive for yellow fever at baseline were excluded. The study protocol was approved by the ethics co- mmittees for studies involving human participants at the Navrongo Health Research Centre, Navrongo, Ghana, and the National Institute for Research in Public Health, Bamako, Mali. All procedures complied with the Declaration of Helsinki. Written informed consent was provided by the participants’ parents or legal guardians in accordance with international ethical guidelines for epidemiological studies and with applicable local ethical guidance and requirements. www.thelancet.com/infection Vol 19 December 2019 Procedures Blood samples were obtained by standard venipuncture. The measured outcomes were the serum concentration of neutralising antibodies to yellow fever virus 4·5 years after vaccination (Mali) or 2·3 and 6·0 years after vaccination (Ghana) and, in each cohort, the relative proportions of seropositive, borderline, and seronegative study participants at these timepoints. Titres of neutralising antibodies to yellow fever virus were determined by a microneutralisation assay. Brieﬂy, 100 50% tissue culture infective doses of a yellow fever virus suspension (strain YF-17D-204, Stamaril, Sanoﬁ Pasteur, Val de Reuil, France) were reacted with serial two-fold dilutions of sera (starting at 1:4) before inoculation into Vero cells cultured in 96-well plates, which were microscopically examined for cytopathic eﬀect after 7 days. Baseline samples were tested in the same run as the matching post-vaccination sera. A neutralisation antibody titre of 1:10 or higher is considered a surrogate of protection.8,9 However, to facilitate comparisons with antibody data collected by others using non-equivalent methods, we converted our titres to standardised concentrations in IU/mL by in- cluding in every assay two standard samples for yellow fever neutralising antibodies, which were them selves calibrated at 426·82 IU/mL and 106·70 IU/mL with reference to the First International Standard for yellow fever vaccine Table 2: Antibody concentration strata for participants with measurable neutralising antibodies (WHO International Standard, NISBC 99/616) recon- stituted at 143·00 IU/mL. On the basis of earlier studies,10–12 we applied a concentration threshold to discriminate seropositive (≥0·5 IU/mL) from borderline sera (measurable concentrations <0·5 IU/mL). Accor dingly, we deﬁned seroconversion and seroreversion as the crossing of this threshold over time. We classiﬁed samples as seronegative if they were ineﬀective at the initial dilution in the assay (1:4). 1365 www.thelancet.com/infection Vol 19 December 2019 Articles Articles signiﬁcance threshold, as estimated by the 95% CI for the p value.14 Further details on the tests are provided in the appendix (p 3). Figure 2: Reverse cumulative distribution plots of neutralising antibody values (A) Raw titres in the plaque reduction seroneutralisation assay for the full study groups. (B) Standardised antibody concentrations for broadly seropositive children (ie, seropositive and borderline study participants). The dotted line denotes the threshold for strict seropositivity at 0·5 IU/ml. Procedures A 0 4 8 16 32 64 128 512 2048 Reciprocal titre 0 20 40 60 80 100 Proportion of participants (%) B 0·125 0·5 1 2 10 100 Antibody concentration (IU/ml) 0 20 40 60 80 100 Proportion of positive and borderline participants (%) Mali, 4·5 years Ghana, 2·3 years Ghana, 6·0 years A 0 4 8 16 32 64 128 512 2048 Reciprocal titre 0 20 40 60 80 100 Proportion of participants (%) Mali, 4·5 years Ghana, 2·3 years Ghana, 6·0 years Mali, 4·5 years Ghana, 2·3 years Ghana, 6·0 years We did statistical analysis with R (version 3.4.3 and version 3.5.3) software. Hypothesis tests were two-sided, and the signiﬁcance threshold was set at α=0·05. Proportion of participants (%) Results We included 436 children (226 [52%] boys, 210 [48%] girls) from rural communities in northern Ghana who received yellow fever vaccination as part of the Expanded Programme on Immunization between June 5, 2009, and Feb 25, 2010, and 587 children (297 [51%] boys, 290 [49%] girls) from urban communities in Mali who received vaccination between March 6, 2012, and Dec 26, 2012. The mean age at which children were vaccinated was 9·0 months (SD 0·3) in Ghana and 9·4 months (0·8) in Mali. The children provided blood samples 2·3 and 6·0 years after vaccination in Ghana and 4·5 years after vaccination in Mali (table 1). Proportion of positive and borderline In the Malian sample, 296 (50·4%; 95% CI 46·4–54·5) of 587 children were seropositive 4·5 years after vaccination (ﬁgure 1). Additionally, we classiﬁed 113 (19·3%, 95% CI 16·1–22·4) children as borderline (antibody concentration <0·5 IU/mL). When we merged positive and borderline strata into a broadly positive category, 409 (69·7%, 95% CI 66·0–73·4; table 2) were seropositive. This proportion presents a substantial drop from the overall number of children who seroconverted (290 [96·7%] of 300 children) 4 weeks after vaccination in data previously reported by others for diﬀerent samples of the same vaccinee population.15 Figure 2: Reverse cumulative distribution plots of neutralising antibody values (A) Raw titres in the plaque reduction seroneutralisation assay for the full study groups. (B) Standardised antibody concentrations for broadly seropositive children (ie, seropositive and borderline study participants). The dotted line denotes the threshold for strict seropositivity at 0·5 IU/ml. Role of the funding source The funders of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. CD and JEM had full access to all the data in the study. The corresponding author had ﬁnal responsibility for the decision to submit for publication. www.thelancet.com/infection Vol 19 December 2019 Statistical analyses By calculating the bounds of the 95% CI by the asymptotic method assuming 50% seropositivity, we determined that a sample of 386 vaccinees or more would constrain the 95% CI for the percentage of seropositive participants to a 5% interval around the empirical percentage. 121 (27·8%; 95% CI 23·5–32·0) of 436 Ghanaian children were seropositive to yellow fever virus 2·3 years after vaccination (figure 1). Among the large majority below the seropositivity threshold, we classified 51 (11·7%, 8·7–14·7) as borderline (table 2). Combination of the positive and borderline strata resulted in 172 children (39·4%, 34·9–44; ﬁgure 1). As in Mali, the proportion of children with broad-sense seropositivity was much smaller than the overall proportion observed 4 weeks after vaccination (611 [72·7%] of 841 children tested) in a previous study of the same Ghanaian population.15 We analysed the two vaccinee samples from Ghana and Mali separately. We used χ² tests to compare the proportions of boys and girls between two immunogenicity strata, Boschloo’s exact unconditional test13 for other 2 × 2 comparisons between independent groups, and McNemar’s test on the proportions for dichotomous outcomes (eg, positives and negatives) in paired sera. We characterised sets of vaccinees by the geometric mean, the median, and the IQR of antibody concen- trations. We used a percentile bootstrap procedure with 10⁶ replications to compute the 95% CI for the geometric mean. We compared two sets of concentrations by either an independent-samples permutation test on the diﬀerence of geometric means or a permutation-based sign test for paired data, as applicable. The permutation tests involved 999 999 random replications, ensuring precision to three decimal digits for the p value near the In summary, despite diﬀerences between our Mali and Ghana groups, these data show a substantial long-term decline in immunity relative to the early outcome of vaccination. We found the prevalence of seropositivity to be greater among the Malian children despite the longer follow-up 1366 Articles time (50·4% [95% CI 46·4–54·5] vs 27·8% [23·5–32·0]; ﬁgure 1, 2A), but the prevalence after 4 weeks from an earlier study15 was also greater in the Malian than in the Ghanaian population samples. Statistical analyses The distributions of measurable antibody concentrations (ie, those of broadly seropositive participants) in the Malian and the year-2·3 Ghanaian sera were similar, and we recorded the same median concentration (0·721 IU/mL), but we observed an excess of higher values in the Ghanaian relative to the Malian sera (ﬁgure 2B; appendix p 4). The geometric mean concentration for the seropositive stratum was accordingly higher in the Ghanaian group at 1·38 IU/mL (95% CI 1·17–1·66) versus 1·12 IU/mL (95% CI 1·04–1·21) in Mali (p=0·0087; table 2; appendix p 5). Figure 3: Evolution of the Ghanaian cohort Flow of study participants from a year-2·3 to a year-6·0 antibody concentration stratum. The thickness of the nodes (initial and ﬁnal) and links between them are proportional to the number of participants as shown on the ﬁgure. Positive, high tier Concentration ≥1·8 IU/ml Positive, low tier Concentration ≥0·5 IU/ml and <1·8 IU/ml Borderline Concentration <0·5 IU/ml Negative Year 2·3 Year 6 11 21 10 23 1 33 40 13 47 13 11 16 24 173 31 90 264 51 78 110 213 35 The paired samples taken 2·3 and 6·0 years after vaccination showed the evolution of immunogenicity in the Ghanaian cohort. This group had a larger proportion of seropositive individuals 6·0 years after vaccination (43·1% [95% CI 38·5–47·8] vs 27·8% [23·5–32·0]; ﬁgure 1; table 2). Year-6·0 antibody concentrations were also higher overall than the Ghana year-2·3 and the Mali year-4·5 values (ﬁgure 2B; appendix p 4). 94 Ghanaian children were serostable over the intervening period, representing 77·7% of the year-2·3 positives and 21·6% of vaccinees (appendix pp 5–6). ( pp pp ) By contrast, 27 seropositive children (22·3% of sero- positive children in year 2·3) seroreverted in the year-6·0 data—ie, deteriorated to negative or bordeline serostatus. These children had markedly lower year-2·3 antibody concentrations than the serostable children (geometric mean 0·74 IU/mL [95% CI 0·67–0·83] vs 1·65 IU/mL [1·35–2·05], p=0·0075; appendix pp 5, 7). We observed that lower year-2·3 concentrations were associated with higher year-6·0 seroreversion frequencies, and that study participants with values 1·8 IU/mL or more at year 2·3 were seroreversion-free (p<0·0001; appendix p 8). We consequently used 1·8 IU/mL as a tentative functional threshold to deﬁne a low and a high tier of seropositives. Statistical analyses 27 (30%) of the 90 low-tier year-2·3 seropositives seroreverted at year 6·0 after vaccination, whereas an even larger proportion of year-2·3 borderline participants (24 [47%] of 51) deteriorated to negative serostatus (ﬁgure 3). Figure 3: Evolution of the Ghanaian cohort Figure 3: Evolution of the Ghanaian cohort Flow of study participants from a year-2·3 to a year-6·0 antibody concentration stratum. The thickness of the nodes (initial and ﬁnal) and links between them are proportional to the number of participants as shown on the ﬁgure. Figure 3: Evolution of the Ghanaian cohort Flow of study participants from a year-2·3 to a year-6·0 antibody concentration stratum. The thickness of the nodes (initial and ﬁnal) and links between them are proportional to the number of participants as shown on the ﬁgure. the geometric mean concentration increased between the two follow-up points in boys, from 1·52 IU/mL (95% CI 1·15–2·05) to 2·18 IU/mL (95% CI 1·66–2·91; p=0·016), but not in girls (appendix pp 6, 10). www.thelancet.com/infection Vol 19 December 2019 Articles The importance of humoral immunity in protecting against yellow fever is well established,6 as is the detection of neutralising antibodies (titre ≥1:10) as a surrogate for protective immunity. The role of T-cell-dependent immunity is uncertain; humoral immunity is known to be crucial, but neither the cellular component nor the presence of neutralising antibodies alone might be suﬃcient for protective immunity.25 The numerous late seroconversions in the Ghanaian year-6·0 data might have arisen from an external cause. The spread of a yellow fever outbreak in the Builsa, Kassena-Nankana West, and Kintampo South districts of Ghana in 2011 overlapped with the catchment area of this study and, chronologically, with the dates of the year-2·3 collection. Additionally, mass vaccination campaigns in the aﬀected areas targeted 5·8 million people in early 2012.35 Vaccine immunity might have been boosted by natural yellow fever virus infection, unreported revaccination, or both. In this scenario, averted antibody decay in some children or new seroconversions in others could lead to an overestimation of the seropositivity resulting from the vaccine received at 9–12 months of age, a common issue in studies of vaccination in endemic areas.6 Unreported infection with wild or vaccinal virus might also have contributed to the enrichment in high antibody concentrations in the Ghanaian relative to the Malian seropositive children, and to the observed increase in year-6·0 antibody values among serostable boys. Although a higher antibody response to yellow fever vaccination has been noted previously in male individuals, the incidence of yellow fever is also greater in male than in female individuals, possibly determined by epidemiological and host-intrinsic factors.36 Additionally, the seroconversion of the majority of borderline participants to the low tier of seropositivity is in line with reports of a negative correlation between pre-existing antibodies against yellow fever and the magnitude of the B-cell response on subsequent exposure to the virus.8,34,37 We normalised assay results to a WHO international standard for yellow fever neutralising antibodies, which makes it possible to compare our data with those from other investigations using standardised antibody concentrations. Additionally, we conservatively introduced a seropositivity cutoﬀ (≥0·5 IU/mL), because very low antibody concentrations might straddle the sensitivity threshold of the assay and were associated with inconsistent protection in previous studies.26,27 Our ﬁndings point to a large decline in yellow fever immunity in children vaccinated as infants. Articles Studies have shown a progressive, long-term decrease in neutralisation titres in adults vaccinated against yellow fever (by 30%–40% within 5–10 years after vaccination),6 but also that children seroconvert at comparatively lower rates and develop weaker antibody titres.17 Waning seroprotection is a major determinant of the need for, and periodicity of, revaccination, and the population immunity achievable with a single dose of vaccine depends on the rate of vaccination failure and on the duration of conferred immunity. These parameters are important for immunisation programmes in endemic areas, where protective immunity covering about 80% of the population is necessary to prevent outbreaks.1 Forecasts of population coverage under the 2017–26 Eliminating Yellow Fever Epidemics strategy,24 which implements vaccination in infants as part of the Expanded Programme on Immu- nization in endemic countries, suggest that targets will not be met if conferred immunity is short-lived. the Expanded Programme on Immunization should be considered as an explanation for the drop in immunity. In some studies, the immunogenicity of the yellow fever vaccine was decreased by co-administration with the measles vaccine, either alone28 or as part of the measles, mumps, rubella combination.29 In other studies,30,38,39 however, no interference could be shown. The sharp deterioration of immunity to yellow fever in our data shows that the eﬀect of infant co-vaccination on the long- term evolution of immunogenicity needs to be addressed speciﬁcally. The diﬀerences in seropositivity between the Ghanaian and Malian groups might owe less to the respective vaccine strains, which are genetically stable,6 than to factors such as vaccine dosage and handling,31 ethnic background,32 nutritional status, a seasonal eﬀect on immunisation,33 exposure to other ﬂaviviruses, or the immune micro- environment.34 A larger proportion of seroconverted individuals was determined for diﬀerent samples from the same study populations (96·7% in Mali and 72·7% in Ghana) 28 days after vaccination in a previous study.15 Relative to the seropositivity in that study (at the ≥1:8 titre threshold), we noted an approximately two times long- term decrease in seropositivity for either population. Direct comparison, however, is hindered by diﬀerences in the population samples and study methods and by non- standardisation of antibody values in that earlier work. We have analysed the long-term persistence of neutralising antibodies against yellow fever virus among two population samples of African children vaccinated under the Expanded Programme on Immunization. www.thelancet.com/infection Vol 19 December 2019 Discussion Since 2013, WHO has recommended a single dose of yellow fever vaccine for life-long immunity, and the amended International Health Regulations (2005) no longer require revaccination every 10 years.5,16 Although it followed evidence of long-term immunogenicity,17,18 this policy change has been controversial.19–22 The one-dose recommendation bypasses the question of the duration of protective immunity elicited in infants, the target population of immunisation programmes in endemic countries.6 Relevant studies have focused on adults or older children, but the duration of immunity against yellow fever following infant vaccination has been studied only over short post-vaccination periods of 1–3 months.23 The scarcity of long-term studies on neutralising antibodies elicited in vaccinated infants has been identiﬁed as a knowledge gap in regard to the need for booster doses of the yellow fever vaccine.23 94 (29·8%) of the 315 year-2·3 seronegative or border- line children seroconverted between the two collection campaigns and accounted for 50% of the seropositive children in year 6·0 (ﬁgure 3; appendix p 7). Negative and borderline children seroconverted in the year-6·0 sample with similar frequencies (30·3% vs 27·0%, p=0·73; ﬁgure 3; appendix p 7). The proportions of low- tier and high-tier seropositive children in each group were, however, very diﬀerent, with seroconversions of borderline participants markedly skewed toward the low tier (p=0·012; ﬁgure 3; appendix p 8). Sex ratios in the immunity strata considered were unbiased relative to the source cohort, and we observed no diﬀerences by sex in antibody values (appendix p 9), except among serostable Ghanaian children, for whom 1367 Articles Articles Articles For anonymised data see https://doi.org/10.5281/ zenodo.2550702 Articles The reduction in seropositivity, especially among the Ghanaian group after only 2·3 years, is noteworthy in comparison with previously reported seroconversion 28 days after vaccination.15 This suggests a rapid waning of immunity during the early years after infant vaccination, similar to the decline suggested to occur in adults during the ﬁrst 5 years6,8 but aﬀecting a greater proportion of individuals or, conceivably, with faster kinetics. Additionally, half of the Ghanaian borderline participants became seronegative at 6·0 years, which suggests that Malian borderline participants (38·8% of borderline and seronegative participants) were likewise aﬀected by waning immunity (eventual seroreversion could not be determined without a later follow-up sample). ) Antigenic interference between the live-virus vaccines against measles and yellow fever co-administered under 1368 Articles The inability to control for environmental factors that might bias the measured response to yellow fever vaccination is a limitation of this study. We cannot exclude immune activation by unrelated infections or immu- nisations within the time frame of sample collection, or an antigenic challenge of memory immunity by other cross- reactive, circulating ﬂaviviruses. Additionally, the presence of other vaccines, notably against measles, prevents the outcome of yellow fever vaccination from being assessed in isolation; however, it is unavoidable in a study in endemic countries with routine immunisation pro- grammes, and the data are useful within this real-life context. The absence of two long-term follow-up points in Mali matching those of the Ghanaian cohort also limits comparisons between the two groups. 3 Garske T, Van Kerkhove MD, Yactayo S, et al. Yellow fever in Africa: estimating the burden of disease and impact of mass vaccination from outbreak and serological data. PLoS Med 2014; 11: e1001638. 4 Shearer FM, Moyes CL, Pigott DM, et al. Global yellow fever vaccination coverage from 1970 to 2016: an adjusted retrospective analysis. Lancet Infect Dis 2017; 17: 1209–17. 5 WHO. Vaccines and vaccination against yellow fever: WHO position paper, June 2013—recommendations. Vaccine 2015; 33: 76–77. 6 Amanna IJ, Slifka MK. Questions regarding the safety and duration of immunity following live yellow fever vaccination. Expert Rev Vaccines 2016; 15: 1519–33. 7 Collaborative Group for Studies of Yellow Fever Vaccination. A randomised double-blind clinical trial of two yellow fever vaccines prepared with substrains 17DD and 17D-213/77 in children nine-23 months old. Mem Inst Oswaldo Cruz 2015; 110: 771–80. 7 Collaborative Group for Studies of Yellow Fever Vaccination. Contributors Contributors CD conceived and designed the study, performed and supervised experimental analyses, and interpreted the results and wrote the manuscript with JEM. JEM also designed and performed the statistical analysis of the data. JF contributed to experimental data collection. POA, NB, CLK, and SOS were responsible for the curation of sample sets and previous relevant data from the MenAfriVac studies. All authors provided critical review and approved the ﬁnal draft of the manuscript. 15 Roy Chowdhury P, Meier C, Laraway H, et al. Immunogenicity of yellow fever vaccine coadministered with MenAfriVac in healthy infants in Ghana and Mali. Clin Infect Dis 2015; 61 (suppl 5): S586–93. 16 WHO. International health regulations (2005), 3rd edn. Geneva: World Health Organization, 2016. 16 WHO. International health regulations (2005), 3rd edn. Geneva: World Health Organization, 2016. 17 Gotuzzo E, Yactayo S, Cordova E. Eﬃcacy and duration of immunity after yellow fever vaccination: systematic review on the need for a booster every 10 years. Am J Trop Med Hyg 2013; 89: 434–44. 17 Gotuzzo E, Yactayo S, Cordova E. Eﬃcacy and duration of immunity after yellow fever vaccination: systematic review on the need for a booster every 10 years. Am J Trop Med Hyg 2013; 89: 434–44. Data sharing 19 Estofolete CF, Nogueira ML. Is a dose of 17D vaccine in the current context of yellow fever enough? Braz J Microbiol 2018; 49: 683–84. The anonymised data have been deposited in Zenodo and can be downloaded from the time of publication. 20 Vasconcelos PF. Single shot of 17D vaccine may not confer life-long protection against yellow fever. Mem Inst Oswaldo Cruz 2018; 113: 135–37. 20 Vasconcelos PF. Single shot of 17D vaccine may not confer life-long protection against yellow fever. Mem Inst Oswaldo Cruz 2018; 113: 135–37. Declaration of interests SOS received grants from PATH during the conduct of this study. SOS is the Minister of Health and Public Hygiene of the Republic of Mali but was not at the time of the collection of the samples used in this study. NB and CK received grants from the Bill and Melinda Gates Foundation during the conduct of this study. All other authors declare no competing interests. 18 Staples JE, Bocchini JA Jr, Rubin L, Fischer M. Yellow fever vaccine booster doses: recommendations of the Advisory Committee on Immunization Practices, 2015. MMWR Morb Mortal Wkly Rep 2015; 64: 647–50. Articles A randomised double-blind clinical trial of two yellow fever vaccines prepared with substrains 17DD and 17D-213/77 in children nine-23 months old. Mem Inst Oswaldo Cruz 2015; 110: 771–80. 8 Hepburn MJ, Kortepeter MG, Pittman PR, et al. Neutralizing antibody response to booster vaccination with the 17D yellow fever vaccine. Vaccine 2006; 24: 2843–49. 8 Hepburn MJ, Kortepeter MG, Pittman PR, et al. Neutralizing antibody response to booster vaccination with the 17D yellow fever vaccine. Vaccine 2006; 24: 2843–49. 9 Monath TP, Cetron MS, McCarthy K, et al. Yellow fever 17D vaccine safety and immunogenicity in the elderly. Hum Vaccin 2005; 1: 207–14. 9 Monath TP, Cetron MS, McCarthy K, et al. Yellow fever 17D vaccine safety and immunogenicity in the elderly. Hum Vaccin 2005; 1: 207–14. p g p Our study shows that the long-term outcome of the vaccination of 9-month-old infants against yellow fever is unsatisfactory and argues for the one-dose-for-life recommendation to be reconsidered for this population in endemic countries. The beneﬁcial eﬀect of a booster dose has been established in individuals with low or negative neutralisation titres,8 but little evidence focuses on children. Further studies will be necessary to plan an ideal revaccination strategy against yellow fever within the wider schedule of childhood immunisations. A booster dose of vaccine would help to attain appropriate population immunity against yellow fever outbreaks. 10 de Menezes Martins R, Maia MLS, de Lima SMB, et al. Duration of post-vaccination immunity to yellow fever in volunteers eight years after a dose-response study. Vaccine 2018; 36: 4112–17. 10 de Menezes Martins R, Maia MLS, de Lima SMB, et al. Duration of post-vaccination immunity to yellow fever in volunteers eight years after a dose-response study. Vaccine 2018; 36: 4112–17. 11 Wieten RW, Goorhuis A, Jonker EFF, et al. 17D yellow fever vaccine elicits comparable long-term immune responses in healthy individuals and immune-compromised patients. J Infect 2016; 72: 713–22. 11 Wieten RW, Goorhuis A, Jonker EFF, et al. 17D yellow fever vaccine elicits comparable long-term immune responses in healthy individuals and immune-compromised patients. J Infect 2016; 72: 713–22. 12 Campi-Azevedo AC, de Almeida Estevam P, Coelho-Dos-Reis JG, et al. Subdoses of 17DD yellow fever vaccine elicit equivalent virological/immunological kinetics timeline. BMC Infect Dis 2014; 14: 391. 13 Lydersen S, Fagerland MW, Laake P. Recommended tests for association in 2×2 tables. Stat Med 2009; 28: 1159–75. 13 Lydersen S, Fagerland MW, Laake P. Articles Recommended tests for association in 2×2 tables. Stat Med 2009; 28: 1159–75. 14 Ruxton GD, Neuhäuser M. Improving the reporting of P-values generated by randomization methods. Methods Ecol Evol 2013; 4: 1033–36. Acknowledgments g The authors are grateful to Ursula Erikli for copy-editing, and to Yuxiao Tang, Cherine Bajali, and Lionel Martellet (Meningitis Vaccine Project, MVP) for excellent guidance and administrative support in facilitating access to the biological samples and accompanying data by agreement with PATH. The samples and previous data were collected in the course of the MenAfriVac trials supported by grants from the Bill and Melinda Gates Foundation (to PATH). Publication costs were paid by the Bill and Melinda Gates Foundation. This work was funded by the Wellcome Trust under the Department for International Development-Wellcome Joint Initiative on Research for Epidemic Preparedness (grant number 206534/Z/17/Z to CD). 21 Grobusch MP, van Aalst M, Goorhuis A. Yellow fever vaccination— once in a lifetime? 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Am J Trop Med Hyg 2002; 66: 533–41. 33 Moore SE, Collinson AC, Fulford AJ, et al. Eﬀect of month of vaccine administration on antibody responses in The Gambia and Pakistan. Trop Med Int Health 2006; 11: 1529–41. 1370 www.thelancet.com/infection Vol 19 December 2019 www.thelancet.com/infection Vol 19 December 2019 Supplementary webappendix This webappendix formed part of the original submission and has been peer reviewed. We post it as supplied by the authors. Supplement to: Domingo C, Fraissinet J, Ansah PO, et al. Long-term immunity against yellow fever in children vaccinated during infancy: a longitudinal cohort study. Lancet Infect Dis 2019; published onlined Sept 19. https://doi.org/10.1016/S1473- 3099(19)30323-8. Contents Appendix note 1: References for the MenAfriVac studies in international trial registries 2 Appendix note 2: Permutation tests 3 Appendix figure 1: Density plots of the distributions of antibody concentrations 4 Appendix figure 2: Box plots of antibody concentrations in categories of seropositive children 5 Appendix table 1: Statistics for serostable participants in the Ghana cohort 6 Appendix table 2: Statistics for seroreverters and late seroconverters in the Ghana cohort 7 Appendix figure 3: Seroreversion and late seroconversion in the Ghanaian group 8 Appendix table 3: Proportions of males and females in the study populations and serological strata 9 Appendix table 4: Mean antibody concentrations by sex and seropositivity tier 9 Appendix table 5: McNemar’s tests on the paired year-2·3 and year-6 sera from the Ghana cohort 10 Appendix references 10 1 1 Appendix note 1: References for the MenAfriVac studies in international trial registries 1. PsATT-004, Ghana. 1. PsATT-004, Ghana. ISRCTN registry number ISRCTN82484612. Independent-samples permutation test We performed a permutation test on two independent antibody concentration sets x and y of size n and m to test the null hypothesis that the geometric mean concentrations (GMCs) are the same between the two groups of study participants. For this, we computed the difference of the respective GMCs, DGMC. Next, we compared this statistic against an empirical null distribution of the difference of GMCs that we generated by the Monte Carlo approach, as follows. We combined x and y into a single dataset (under the null hypothesis that the two sets of observations are identically distributed), drew a randomly-permuted dataset of size N = n + m by sampling the combined dataset without replacement, and split this permuted dataset into two subsets x' and y' of size n and m. We computed and recorded the difference of GMCs between x' and y', DGMC’, and repeated the sampling and computation steps for B = 999999 total replications. Lastly, we computed a p-value by the formula: ! = ($ + 1) (( + 1) ⁄ (1) ! = ($ + 1) (( + 1) ⁄ (1) where T denotes the number of cases in the permutation distribution where DGMC’ ≥ DGMC in absolute value (two-tailed test). denotes the number of cases in the permutation distribution where DGMC’ ≥ DGMC in absolute value ed test). The permutation tests were designed after Good.1 The R scripts implementing these tests are available at the URL: https://doi.org/10.5281/zenodo.2684194 The permutation tests were designed after Good.1 The R scripts implementing these tests are available at the URL: https://doi.org/10.5281/zenodo.2684194 Paired-samples permutation test We performed a paired-samples permutation test to assess net changes in antibody concentrations between the first and second serum collections from the same study group of size n. Specifically, we tested the null hypothesis that the sum of differences between the paired concentrations equaled zero. We log-transformed the concentration data, and computed the sum of differences S across the n pairs of values. Next, we took the absolute value of each difference, gave it a plus or minus sign at random, and computed and recorded the sum of the n randomly-signed differences, S’. We repeated this step for B = 999999 total replications to build an empirical null distribution for the sum of differences. Lastly, we computed a p-value using equation (1) above with T as the number of cases in the permutation distribution where S’ ≥ S in absolute value (two-tailed test). Choice of the number of permutations URL: https://doi.org/10.1186/ISRCTN82484612 URL: https://doi.org/10.1186/ISRCTN82484612 URL: https://doi.org/10.1186/ISRCTN82484612 A phase II, double-blind, randomised, controlled, dose ranging study to evaluate the safety, immunogenicity, dose response and schedule response of a meningococcal A conjugate vaccine administered concomitantly with local expanded program on immunisation (EPI) vaccines in healthy infants. Pers-007 (phase IV), Ghana. ISRCTN registry number ISRCTN10763234. URL: https://doi.org/10.1186/ISRCTN10763234 . Pers-007 (phase IV), Ghana. ISRCTN registry number ISRCTN10763234. URL: https://doi.org/10.1186/ISRCTN10763234 2. Pers-007 (phase IV), Ghana. ISRCTN registry number ISRCTN10763234. URL: https://doi.org/10.1186/ISRCTN10763234 Evaluation of antibody persistence in Ghanaian children more than five years after vaccination with MenAfriVac® widely used in Sub-Saharan Africa to prevent epidemic meningitis. 3. PsATT-007, Mali. Pan African Clinical Trials Registry number PACTR201110000328305 URL: https://pactr.samrc.ac.za/TrialDisplay.aspx?TrialID=328 3. PsATT-007, Mali. Pan African Clinical Trials Registry number PACTR201110000328305 URL: https://pactr.samrc.ac.za/TrialDisplay.aspx?TrialID=328 3. PsATT-007, Mali. Pan African Clinical Trials Registry number PACTR201110000328305 , Pan African Clinical Trials Registry number PACTR201110000328305 URL: https://pactr.samrc.ac.za/TrialDisplay.aspx?TrialID=328 A Phase III, double-blind, randomized, controlled study to evaluate the immunogenicity and safety of different schedules and formulations of a meningococcal A conjugate vaccine administered concomitantly with local EPI vaccines in healthy infants and toddlers. 4. Pers-007 (phase IV), Mali. ISRCTN registry number ISRCTN37623829 URL: https://doi.org/10.1186/ISRCTN37623829 4. Pers-007 (phase IV), Mali. g y URL: https://doi.org/10.1186/ISRCTN37623829 ong-term follow-up of children who participated at 9-12 months of age in clinical trial PsA-TT-007 in Ma Long-term follow-up of children who participated at 9-12 months of age in clinical trial PsA-TT-007 in 2 2 Appendix note 2: Permutation tests Appendix note 2: Permutation tests The permutation tests were designed after Good.1 The R scripts implementing these tests are available at the URL: https://doi.org/10.5281/zenodo.2684194 Choice of the number of permutations B = 999999 was selected as sufficient to provide precision to three decimal digits when p = 0·05, i.e. such that the 95% confidence interval (CI) for the true p-value lie inside the interval [0·0495, 0·0505]. The bounds of the 95% CI for the true p-value were computed by the following formula after Ruxton and Neuhäuser,2 ! ± +(,-.·01)23(,-3) 45, (2) (2) where z(1 − 0·5a) denotes the (1 − 0·5a) quantile of the standard normal distribution (≈ 1·96 for a = 0·05), and p = 0·05. 3 Antibody concentration (IU/ml) Density 0·1 0·5 1 1·8 10 100 0 0·1 0·2 0·3 0·4 Mali, 4·5 y Ghana, 2·3 y Ghana, 6 y Appendix figure 1: Kernel density plot of the distributions of antibody concentrations. The figure compares the distributions of antibody concentrations between study groups and follow- up time points. The data correspond in each case to the combined seropositive and borderline strata. The dotted lines denote the seropositivity threshold (0·5 IU/ml), and the 1·8 IU/ml threshold between the low and high tiers of seropositives. Antibody concentration (IU/ml) Density 0·1 0·5 1 1·8 10 100 0 0·1 0·2 0·3 0·4 Mali, 4·5 y Ghana, 2·3 y Ghana, 6 y Density Antibody concentration (IU/ml) Appendix figure 1: Kernel density plot of the distributions of antibody concentrations. The figure compares the distributions of antibody concentrations between study groups and follow- up time points. The data correspond in each case to the combined seropositive and borderline strata. The dotted lines denote the seropositivity threshold (0·5 IU/ml), and the 1·8 IU/ml threshold between the low and high tiers of seropositives. Choice of the number of permutations 4 p = 0·0075 p = 0·12 p = 0·28 p = 1 × 106 p 1 p = 0·20 p = 0·0081 p = 0·0087 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● 0.5 1 2 4 8 16 32 64 Antibody concentration (IU/ml) Mali, year-4·5 sera total positives n = 296 Ghana, year-2·3 sera total positives n = 121 Ghana, year-6 sera total positives n = 188 Ghana, year-2·3 sera seroreverters at year 6 n = 27 Ghana, year-2·3 sera serostable at year 6 n = 94 Ghana, year-6 sera serostable since year 2·3 n = 94 Ghana, year-6 sera late seroconverters n = 94 Appendix figure 2: Box plots of antibody concentrations in categories of seropositive children. White dots denote the respective geometric mean concentrations (GMCs); paired data sets are connected by an underside bracket. Two-tailed p-values test for equality of GMCs (independent-samples permutation test) or a net concentration change in the paired samples (permutation sign-test). Appendix figure 2: Box plots of antibody concentrations in categories of seropositive children. White dots denote the respective geometric mean concentrations (GMCs); paired data sets are connected by an underside bracket. Two-tailed p-values test for equality of GMCs (independent-samples permutation test) or a net concentration change in the paired samples (permutation sign-test). 5 I. Serostable participants N Percentage of vaccinees (95% CI) Percentage of year-2·3 seropositives Percentage of year-6 seropositives Males (%) Females (%) Total 94 21·6% (17·7–25·4) 77·7% 50·0% 49 (52%) 45 (48%) Low tier, year 2·3 63 14·4 52·1% n.a. 36 (57%) 27 (43%) High tier, year 2·3 31 7·1 25·6% n.a. 13 (42%) 18 (58%) Low tier, year 6 50 11·5 n.a. 26·6% 22 (44%) 28 /56%) High tier, year 6 44 10·1 n.a. Choice of the number of permutations 23·4% 27 (61%) 17 (39%) II. Antibody concentrations (IU/ml) GMC (95% CI) Median concentration (IQR) GMC, males (95% CI) GMC, females (95% CI) Year-2·3 sera Total 1·65 (1·35–2·05) 1·29 (0·762–2·87) 1·52 (1·15–2·05) 1·81 (1·35–2·50) Low tier 0·908 (0·827–0·997) 0·858 (0·684–1·29) 0·906 (0·798–1·03) 0·911 (0·797–1·04) High tier 5·56 (4·12–7·78) 4·08 (2·89–8·16) 6·33 (3·97–10·4) 5·07 (3·50–8·07) Year-6 sera Total 1·94 (1·60–2·38) 1·60 (0·873–2·89) 2·18 (1·66–2·91) 1·71 (1·31–2·27) Low tier 0·931 (0·844–1·03) 0·910 (0·721–1·26) 0·920 (0·800–1·06) 0·940 (0·819–1·08) High tier 4·47 (3·57–5·68) 3·41 (2·04–8·14) 4·41 (3·28–6·05) 4·58 (3·27–6·63) Appendix table 1: Statistics for serostable participants in the Ghana cohort. The data paired by dotted-line brackets show an increase in antibody concentrations in year 6 among males but not among females. The low and high tiers of seropositive participants are differentiated at the ≥1·8 IU/ml threshold. CI, confidence interval; GMC, geometric mean concentration; IQR, interquartile range; n.a., not applicable. Appendix table 1: Statistics for serostable participants in the Ghana cohort. The data paired by dotted-line brackets show an increase in antibody concentrations in year 6 among males but not among females. The low and high tiers of seropositive participants are differentiated at the ≥1·8 IU/ml threshold. CI, confidence interval; GMC, geometric mean concentration; IQR, interquartile range; n.a., not applicable. 6 6 I. Seroreversion N Percentage of vaccinees (95% CI) Percentage of year- 2·3 seropositives Percentage of year-6 total seronegatives From the low tier of seropositives From the high tier of seropositives Males (%) Females (%) Total seroreverters 27 6·2% 22·3% 10·9% 27 0 17 (63%) 10 (37%) To seronegative 16 3·7% 13·2% 6·5% 16 0 10 (62%) 6 (38%) To borderline 11 2·5% 9·1% 4·4% 11 0 7 (64%) 4 (36%) Year-2·3 antibody concentrations (IU/ml) GMC (95% CI) Median IQR Total seroreverters 0·741 (0·665–0·832) 0·644 0·605–0·915 From the low tier 0·741 (0·665–0·832) 0·644 0·605–0·915 From the high tier n.a. n.a. n.a. To seronegative 0·689 (0·613–0·778) 0·610 0·591–0·907 To borderline 0·823 (0·686–1·01) 0·762 0·607–0·965 II. Choice of the number of permutations Late seroconversion N Percentage of vaccinees (95% CI) Percentage of year- 2·3 non-seropositives Percentage of year-6 total seropositives From seronegative (percentage of seronegatives) From bordeline (percentage of borderline) Males (%) Females (%) Total seroconverters 94 21·6% 29·8% 50·0% 80 (30·3%) 14 (27%) 50 (53%) 44 (47%) To the low tier 60 13·8% 19·0% 31·9% 47 (17·8%) 13 (25%) 36 (60%) 24 (40%) To the high tier 34 7·8% 10·8% 18·1% 33 (12·5%) 1 (2%) 14 (41%) 20 (59%) Year-6 antibody concentrations (IU/ml) GMC (95% CI) Median IQR Total seroconverters 1·65 (1·35–2·06) 1·28 0·765–2·43 From seronegative 1·75 (1·40–2·24) 1·44 0·832–2·86 From borderline 1·16 (0·838–1·90) 1·02 0·763–1·23 To the low tier 0·901 (0·824–0·986) 0·911 0·643–1·19 To the high tier 4·80 (3·49–6·92) 2·88 2·37–5·77 Appendix table 2: Statistics for seroreverters and late seroconverters in the Ghana cohort. Seronegative denotes no read-out at the lowest dilution in the assay. Borderline, measurable antibody concentration <0·5 IU/ml. Seropositive, antibody concentration ≥0·5 IU/ml. Seroreversion denotes deterioration to the borderline or negative stratum; late seroconversion denotes amelioration of negative or borderline participants to the seropositive stratum 6 years postvaccination. The low and high tiers of seropositive participants are differentiated at the ≥1·8 IU/ml threshold. CI, confidence interval; GMC, geometric mean concentration; IQR, interquartile range; n.a., not applicable. Appendix table 2: Statistics for seroreverters and late seroconverters in the Ghana cohort. Seronegative denotes no read-out at the lowest dilution in the assay. Borderline, measurable antibody concentration <0·5 IU/ml. Seropositive, antibody concentration ≥0·5 IU/ml. Seroreversion denotes deterioration to the borderline or negative stratum; late seroconversion denotes amelioration of negative or borderline participants to the seropositive stratum 6 years postvaccination. The low and high tiers of seropositive participants are differentiated at the ≥1·8 IU/ml threshold. CI, confidence interval; GMC, geometric mean concentration; IQR, interquartile range; n.a., not applicable. Appendix table 2: Statistics for seroreverters and late seroconverters in the Ghana cohort. Seronegative denotes no read-out at the lowest dilution in the assay. Borderline, measurable antibody concentration <0·5 IU/ml. Seropositive, antibody concentration ≥0·5 IU/ml. Seroreversion denotes deterioration to the borderline or negative stratum; late seroconversion denotes amelioration of negative or borderline participants to the seropositive stratum 6 years postvaccination. The low and high tiers of seropositive participants are differentiated at the ≥1·8 IU/ml threshold. CI, confidence interval; GMC, geometric mean concentration; IQR, interquartile range; n.a., not applicable. Appendix table 2: Statistics for seroreverters and late seroconverters in the Ghana cohort. Choice of the number of permutations Seronegative denotes no read-out at the lowest dilution in the assay. Borderline, measurable antibody concentration <0·5 IU/ml. Seropositive, antibody concentration ≥0·5 IU/ml. Seroreversion denotes deterioration to the borderline or negative stratum; late seroconversion denotes amelioration of negative or borderline participants to the seropositive stratum 6 years postvaccination. The low and high tiers of seropositive participants are differentiated at the ≥1·8 IU/ml threshold. CI, confidence interval; GMC, geometric mean concentration; IQR, interquartile range; n.a., not applicable. Appendix table 2: Statistics for seroreverters and late seroconverters in the Ghana cohort. Seroneg 7 B Initial classication at year 2·3 of late seroconverters Borderline Negative 100 p = 0·012 0·5 1 1·8 10 Antibody concentration (IU/ml) 6 years after vaccination 1 33 13 47 Appendix figure 3: Seroreversion and late seroconversion in the Ghanaian group. Seroreversion denotes deterioration to the borderline or negative stratum; late seroconversion denotes amelioration of negative or borderline participants to the seropositive stratum 6 years postvaccination. (A) Percentage of seroreverters as a function of a moving concentration threshold. The 1·8-IU/ml threshold (dotted line) defined a seroreversion-free high tier of seropositive participants. (B) Year-6 antibody concentrations for late seroconverters (n=94), split by year-2·3 source stratum; p-value from Boschloo's test of the proportions of low- and high-tier seropositives (inset).- B Initial classication at year 2·3 of late seroconverters Borderline Negative 100 p = 0·012 0·5 1 1·8 10 Antibody concentration (IU/ml) 6 years after vaccination 1 33 13 47 A 0·5 1 4 1·8 Antibody concentration (IU/ml) 2·3 years after vaccination 95% CI 0 20 30 10 Percentage of seroreversion Low tier High tier A B 0·5 1 4 1·8 Antibody concentration (IU/ml) 2·3 years after vaccination 95% CI 0 20 30 10 Percentage of seroreversion Low tier High tier Percentage of seroreversion Antibody concentration (IU/ml) 2·3 years after vaccination Appendix figure 3: Seroreversion and late seroconversion in the Ghanaian group. Seroreversion denotes deterioration to the borderline or negative stratum; late seroconversion denotes amelioration of negative or borderline participants to the seropositive stratum 6 years postvaccination. (A) Percentage of seroreverters as a function of a moving concentration threshold. The 1·8-IU/ml threshold (dotted line) defined a seroreversion-free high tier of seropositive participants. Choice of the number of permutations (B) Year-6 antibody concentrations for late seroconverters (n=94), split by year-2·3 source stratum; p-value from Boschloo's test of the proportions of low- and high-tier seropositives (inset).- 8 8 Number of participants Study population Total Males (%) Females (%) P-value c2 test Mali, year 4·5 587 297 (50·6%) 290 (49·4%) n.a. n.a. Ghana, year 2·3 and year 6 436 226 (51·8%) 210 (48·2%) n.a. n.a. A. Seropositive participants (antibody concentration ≥0·5 IU/ml) Mali, year 4·5 Total 296 151 (51%) 145 (49%) 0·91 1 Low tier 242 127 (52·5%) 115 (47·5%) 0·29 2 High tier 54 24 (44%) 30 (56%) Ghana, year 2·3 Total 121 66 (54·5%) 55 (45·5%) 0·58 1 Low tier 90 53 (59%) 37 (41%) 0·14 2 High tier 31 13 (42%) 18 (58%) Ghana, year 6 Total 188 99 (52·7%) 89 (47·3%) 0·83 1 Low tier 110 58 (52·7%) 52 (47·3%) 1 2 High tier 78 41 (53%) 37 (47%) B. Borderline participants (measurable antibody concentration <0·5 IU/ml) Mali, year 4·5 113 52 (46%) 61 (54%) 0·35 1 Ghana, year 2·3 51 27 (53%) 24 (47%) 0·89 1 Ghana 6 years 35 18 (51%) 17 (49%) 1 1 C = A + B. Broadly seropositive participants (all participants with a measurable antibody concentration) Mali, year 4·5 409 203 (49·6%) 206 (50·4%) 0·74 1 Ghana, year 2·3 172 93 (54·1%) 79 (45·9%) 0·59 1 Ghana, year 6 223 117 (52·5%) 106 (47·5%) 0·89 1 Appendix table 3: Proportions of males and females in the study populations and serological strata. c2 test 1: Tests the hypothesis that the proportions of males and females in a given serological stratum are the same as in the total study population. c2 test 2: Tests the hypothesis that the proportions of low- and high-tier seropositives are equal between males and females. The low and high tiers of seropositive participants are differentiated at the ≥1·8 IU/ml threshold. n.a., not applicable. Number of participants Number of participants Appendix table 3: Proportions of males and females in the study populations and serological strata. c2 test 1: Tests the hypothesis that the proportions of males and females in a given serological stratum are the same as in the total study population. c2 test 2: Tests the hypothesis that the proportions of low- and high-tier seropositives are equal between males and females. The low and high tiers of seropositive participants are differentiated at the ≥1·8 IU/ml threshold. Choice of the number of permutations n.a., not applicable. Appendix table 3: Proportions of males and females in the study populations and Appendix table 3: Proportions of males and females in the study populations and serological strata. c2 test 1: Tests the hypothesis that the proportions of males and females in a given serological stratum are the same as in the total study population. c2 test 2: Tests the hypothesis that the proportions of low- and high-tier seropositives are equal between males and females. The low and high tiers of seropositive participants are differentiated at the ≥1·8 IU/ml threshold. n.a., not applicable. Appendix table 3: Proportions of males and females in the study populations and serological strata. c2 test 1: Tests the hypothesis that the proportions of males and females in a given serological stratum are the same as in the total study population. c2 test 2: Tests the hypothesis that the proportions of low- and high-tier seropositives are equal between males and females. The low and high tiers of seropositive participants are differentiated at the ≥1·8 IU/ml threshold. n.a., not applicable. Geometric mean concentration and 95% CI (IU/ml) Study population Males Females P-value Seropositive participants (antibody concentration ≥0·5 IU/ml) Mali, year 4·5 1·06 (0·964–1·17) 1·18 (1·05–1·33) 0·16 Low tier 0·858 (0·808–0·912) 0·869 (0·815–0·926) 0·79 High tier 3·20 (2·78–3·73) 3·82 (3·12–4·77) 0·23 Ghana, year 2·3 1·28 (1·03–1·62) 1·52 (1·17–2·02) 0·35 Low tier 0·863 (0·779–0·957) 0·842 (0·755–0·942) 0·77 High tier 6·33 (3·97–10·4) 5·07 (3·50–8·06) 0·51 Ghana, year 6 1·80 (1·46–2·24) 1·78 (1·47–2·18) 0·95 Low tier 0·876 (0·802–0·959) 0·960 (0·869–1·06) 0·19 High tier 4·97 (3·76–6·78) 4·25 (3·31–5·59) 0·45 Appendix table 4: Mean antibody concentrations by sex and seropositivity tier. The low and high tiers of seropositive participants are differentiated at the ≥1·8 IU/ml threshold. P-value from an independent-samples permutation test on the difference of geometric mean concentrations. Geometric mean concentration and 95% CI (IU/ml) Appendix table 4: Mean antibody concentrations by sex and seropositivity tier. The low and high tiers of seropositive participants are differentiated at the ≥1·8 IU/ml threshold. P-value from an independent-samples permutation test on the difference of geometric mean concentrations. Appendix table 4: Mean antibody concentrations by sex and seropositivity tier. The low and high tiers of seropositive participants are differentiated at the ≥1·8 IU/ml threshold. P-value from an independent-samples permutation test on the difference of geometric mean concentrations. Choice of the number of permutations Cohort-wide seroconversion versus seroreversion events between years 2·3 and 6 post-vaccination. Seroconversion exceeds seroreversion. B. Cohort-wide amelioration versus deterioration cases between years 2·3 and 6 post-vaccination. Similar to (A) above but merging together the seropositive and borderline strata. Amelioration exceeds deterioration. C. Serostable females: upward transitions from the low to the high tier of seropositives, versus downward transitions from the high to the low tier, between years 2·3 and 6 post-vaccination. Upward and downward transitions are approximately balanced. D. Serostable males: upward transitions from the low to the high tier of seropositives, versus downward transitions from the high to the low tier, between years 2·3 and 6 post-vaccination. Upward transitions exceed downward transitions. D. Serostable males: upward transitions from the low to the high tier of seropositives, versus downward transitions from the high to the low tier, between years 2·3 and 6 post-vaccination. Upward transitions exceed downward transitions. 1. Good P. Permutation tests: a practical guide to resampling methods for testing hypotheses. 2nd ed. New York: Springer-Verlag, 2000. Choice of the number of permutations 9 Two-sided McNemar’s test Comparison Number of participants p-value Odds ratio (95% CI) A 7 × 10-10 3·48 (2·25–5·56) Year 6 Year 2·3 Seronegative and borderline Seropositive Seronegative and borderline 221 94 Seropositive 27 94 B 1 × 10-5 2·28 (1·55–3·39) Year 6 Year 2·3 Seronegative Seropositive and borderline Seronegative 173 91 Seropositive and borderline 40 132 C 1 0·875 (0·27–2·76) Females, year 2·3 Females, year 6 Low tier High tier Low tier 20 7 High tier 8 10 D 0·0013 8 (1·88–71·7) Males, year 6 Males, year 2·3 Low tier High tier Low tier 20 16 High tier 2 11 Appendix table 5: McNemar’s tests on the paired year-2·3 and year-6 sera from the Ghana cohort. Seronegative denotes no read-out at the lowest dilution in the seroneutralization assay. Borderline, measurable antibody concentration <0·5 IU/ml. Seropositive, antibody concentration ≥0·5 IU/ml. The low and high tiers of seropositive participants are differentiated at the ≥1·8 IU/ml threshold. CI, confidence interval. A. Cohort-wide seroconversion versus seroreversion events between years 2·3 and 6 post-vaccination. Seroconversion exceeds seroreversion. Number of participants Number of participants Comparison A 7 × 10-10 3·48 (2·25–5·56) Year 6 Year 2·3 Seronegative and borderline Seropositive Seronegative and borderline 221 94 Seropositive 27 94 B 1 × 10-5 2·28 (1·55–3·39) Year 6 Year 2·3 Seronegative Seropositive and borderline Seronegative 173 91 Seropositive and borderline 40 132 C 1 0·875 (0·27–2·76) Females, year 2·3 Females, year 6 Low tier High tier Low tier 20 7 High tier 8 10 D 0·0013 8 (1·88–71·7) Males, year 6 Males, year 2·3 Low tier High tier Low tier 20 16 High tier 2 11 Appendix table 5: McNemar’s tests on the paired year-2·3 and year-6 sera from the Ghana cohort. Seronegative denotes no read-out at the lowest dilution in the seroneutralization assay. Borderline, measurable antibody concentration <0·5 IU/ml. Seropositive, antibody concentration ≥0·5 IU/ml. The low and high tiers of seropositive participants are differentiated at the ≥1·8 IU/ml threshold. CI, confidence interval. Appendix table 5: McNemar’s tests on the paired year-2·3 and year-6 sera from the Ghana cohort. Seronegative denotes no read-out at the lowest dilution in the seroneutralization assay. Borderline, measurable antibody concentration <0·5 IU/ml. Seropositive, antibody concentration ≥0·5 IU/ml. The low and high tiers of seropositive participants are differentiated at the ≥1·8 IU/ml threshold. CI, confidence interval. seropositive participants are differentiated at the ≥1·8 IU/ml threshold. CI, confidence interval. A. Appendix references 1. Good P. Permutation tests: a practical guide to resampling methods for testing hypotheses. 2nd ed. New York: Springer-Verlag, 2000. 2. Ruxton GD, Neuhäuser M. Improving the reporting of P-values generated by randomization methods. Methods Ecol Evol 2013; 4: 1033-6. 2. Ruxton GD, Neuhäuser M. Improving the reporting of P-values generated by randomization methods. Methods Ecol Evol 2013; 4: 1033-6. 10
https://openalex.org/W1985480318	https://link.springer.com/content/pdf/10.1007/s10067-014-2571-9.pdf	English	null	Reactive oxygen species and serum antioxidant defense in juvenile idiopathic arthritis	Clinical rheumatology	2,014	cc-by	3,886	Reactive oxygen species and serum antioxidant defense in juvenile idiopathic arthritis Joanna Lipińska & Stanisława Lipińska & Jerzy Stańczyk & Agata Sarniak & Anna Przymińska vel Prymont & Marek Kasielski & Elżbieta Smolewska Received: 13 January 2014 /Revised: 1 March 2014 /Accepted: 2 March 2014 /Published online: 22 March 2014 # The Author(s) 2014. This article is published with open access at Springerlink.com Received: 13 January 2014 /Revised: 1 March 2014 /Accepted: 2 March 2014 /Published online: 22 March 2014 # The Author(s) 2014. This article is published with open access at Springerlink.com Abstract In autoimmune inflammatory diseases, including juvenile idiopathic arthritis (JIA), which leads to joint destruc- tion, there is an imbalance between production of reactive oxygen species (ROS) and their neutralization which, as a consequence, leads to “oxidative stress.” The aim of the study was to assess the concentration of oxidative stress markers: nitric oxide (NO), a degree of lipid membrane damage, and total antioxidant plasma capacity in children with JIA. Thirty- four children with JIA were included into the study. A degree of lipid membrane damage (lipid peroxidation products) was estimated as thiobarbituric acid-reactive substances (TBARs), NO concentration as NO end-products: nitrite/nitrate (NO2 −/NO3 −) and total antioxidant plasma capacity as ferric reducing ability of plasma (FRAP). NO2 −/NO3 −serum con- centration in children with JIA was statistically significantly higher than that in healthy children (p=0.00069). There was no significant difference in TBAR levels between children with JIA and the control group. FRAP in sera of children with JIA was lower than that in healthy children, but the difference was not statistically significant. A statistically significant positive correlation was observed between NO end products and the 27-joint juvenile arthritis disease activity score (JADAS-27) and ESR, and a negative correlation was ob- served between FRAP and C-reactive protein (CRP) and white blood cell count (WBC). Our results confirm the in- creased oxidative stress in children with JIA. Overproduction of NO and decrease in the antioxidant plasma capacity may be involved in JIA pathogenesis. Keywords Ferric reducing ability of plasma (FRAP) . Juvenile idiopathic arthritis (JIA) . Nitrates (NO3 −) . Nitric oxide (NO) . Nitrites (NO2 −) . Thiobarbituric acid-reactive substances (TBARs) Keywords Ferric reducing ability of plasma (FRAP) . Clin Rheumatol (2015) 34:451–456 DOI 10.1007/s10067-014-2571-9 Clin Rheumatol (2015) 34:451–456 DOI 10.1007/s10067-014-2571-9 ORIGINAL ARTICLE J. Lipińska (): J. Stańczyk: E. Smolewska Department of Pediatric Cardiology and Rheumatology, Second Chair of Pediatrics, Medical University of Lodz, 36/50 Sporna St, 91-738 Lodz, Poland e-mail: joanna-lipinska@wp.pl Introduction Juvenile idiopathic arthritis (JIA) is a heterogeneous group of autoimmune diseases of unknown origin, with onset before the age of 16 years. JIA is considered to be the most common connective tissue disease of childhood. It is characterized by leukocyte infiltration in the synovium leading to a chronic inflammation in the joints, which persists for more than 6 weeks, with consequent destruction of articular tissue [1, 2]. Recently, considerable interest has been generated in determining the JIA pathogenesis, looking for new therapeutic strategies and for the prognostic factors, which would permit an early identification of patients with a poor prognosis, justifying the application of an aggressive treatment in the early stages of the disease [2, 3]. J. Lipińska (): J. Stańczyk: E. Smolewska Department of Pediatric Cardiology and Rheumatology, Second Chair of Pediatrics, Medical University of Lodz, 36/50 Sporna St, 91-738 Lodz, Poland e-mail: joanna-lipinska@wp.pl S. Lipińska: A. Sarniak: A. Przymińska vel Prymont Department of General Physiology, Chair of Experimental and Clinical Physiology, Medical University of Lodz, Mazowiecka 6/8, 92-215 Lodz, Poland Reactive oxygen species (ROS) play a pivotal role in phys- iological processes. ROS mediate and regulate many cell func- tions, including cytokine production, signal transduction, mi- tochondrial functions, immune processes, gene expression, and apoptosis. The effects of ROS depend on their serum concen- tration. Nitric oxide (NO), a unique biological messenger E. Smolewska Outpatient Department of Pediatric Rheumatology, Maria Konopnicka’ Memorial Hospital Lodz, Lodz, Poland E. Smolewska Data are means ± standard deviation (SD), unless otherwise indicated n number of children, ERS erythrocyte sedimentation ratio, CRP C-reactive protein Data are means ± standard deviation (SD), unless otherwise indicated n number of children, ERS erythrocyte sedimentation ratio, CRP C-reactive protein The aim of the study was to assess markers of the oxidative stress: concentrations of NO end-products nitrite/nitrate (NO2 −/NO3 −), thiobarbituric acid-reactive substances (TBARs), and total antioxidant capacity of plasma via ferric reducing ability of plasma (FRAP) in the sera of children with JIA in comparison with healthy children. Moreover, the study also aimed to correlate these markers with disease character- istics (type of JIA onset, disease activity). Serum samples (3 mL) were obtained simultaneously with routine laboratory tests (white blood cell (WBC) count, red blood cell (RBC) count, thrombocyte count, hemoglobin, C- reactive protein (CRP)). The activity of the rheumatoid pro- cess was assessed according to the 27-joint juvenile arthritis disease activity score (JADAS-27) [11]. Each patient was evaluated for the physician’s global assessment of overall disease activity, which was measured on a 10-cm visual analog scale (VAS; 0=no activity and 10=maximum activity); parent’s global assessment of the child’s overall well-being, which was measured on a 10-cm VAS (0=very good and 10= very poor); and 27-joint count including the cervical spine, elbows, wrists, metacarpophalangeal joints (from the first to the third), proximal interphalangeal joints, hips, knees, ankles, and ESR. Patients Thirty-four children (22 girls, 12 boys) with JIAwere included into the study. The patients were entered in regard to the 2001 International League Against Rheumatism (ILAR) classifica- tion criteria [10]. Twenty-six children had polyarticular JIA type of onset, and eight had oligoarticular JIA. They were aged 7–18 years (mean 13.8±1.2 years, range 7–18), had disease duration time of 0.5–7.5 years (mean 3.5±0.8 years), and were treated with DMARDs (methotrexate, 28 patients or sulfasalazine, 6 patients) and low doses of steroids (predni- sone <10 mg/day). All of the study group children were biologically naive (Table 1). The study was approved by the local ethical committee. In every case, a written informed consent was obtained from the patient and parents before study entry. E. Smolewska M. Kasielski Center of Medical Education, Practical Training Center, Medical University of Lodz, Lodz, Poland 452 Clin Rheumatol (2015) 34:451–456 Table 1 Demographic, clinical, and laboratory parameters of children with JIA Characteristics of children with JIA (n=34) Female/male, n (%) 22/12 (64.7/35.3) Age (years)/range 13.8±1.2/7–18 Type of JIA onset Polyarticular, n (%) 26 (76.5) IgM-RF-negative 24 IgM-RF-positive 2 Oligoarticular, n (%) 8 (23.5) Persistent oligoarticular JIA 7 Extended oligoarticular JIA 1 Disease duration time (years) 3.5±0.8 ESR (mm/h) 19.4±20.5 (median 10) ESR above cutoff value (>20 mm/h), n (%) 8 (23.5) CRP (mg/dL) 2.67±6.26 (median 0.39) CRP above cutoff value (>0.5 mg/dL), n (%) 14 (41.2) IgM-RF positivity (≥24 RU/mL), n (%) 2 (5.9) Disease activity JADAS-27 23.94±10.10 (median 25) Data are means ± standard deviation (SD), unless otherwise indicated n number of children, ERS erythrocyte sedimentation ratio, CRP C-reactive protein Table 1 Demographic, clinical, and laboratory parameters of children with JIA molecule, is a short-lived gaseous free radical. It is synthesized from L-arginine by NO synthase in various cell types such as chondrocytes, synoviocytes, and leukocytes, e.g., phagocytic neutrophils. Direct measurement of NO and other free radicals in standard laboratories is difficult because of their biochemical instability. Due to the above, concentrations of NO end-prod- ucts nitrite/nitrate (NO2 −/NO3 −) or the activity of NO synthase is usually evaluated. NO is involved in immune regulation, neurotransmission, and vasodilatation. Recently, the imbal- ance between formation of ROS and body antioxidant de- fense in homeostasis disturbances is widely discussed [4]. The currently reported data demonstrated a significant role of ROS in the etiopathogenesis of autoimmune diseases. In chronic inflammatory processes, after long stimulation, the exhaustion of the body’s antioxidative reserves results in a harmful activity of ROS, as they gain an advantage over the antioxidant system—this phenomenon is called “oxidative stress” [5]. Characteristics of children with JIA (n=34) Many studies are conducted on the role of ROS in the etiopathogenesis of rheumatoid arthritis (RA), which seem to be a main direct factor destroying the joint tissues, but in the pediatric population with JIA, their role has still not been elucidated [5–9]. It could be hypothesized that patients with JIA have defective defense mechanisms against ROS and these mechanisms vary according to the JIA subtypes. FRAP assay The total antioxidant plasma capacity was assayed in terms of the ferric reducing ability of plasma (FRAP) according to the original description by Benzie and Strain [14]. This method measures the ability of antioxidants contained in a sample to reduce ferric tripyridyltriazine (Fe3+-TPTZ) to a ferrous form (Fe2+-TPTZ) that absorbs light at λ=593 nm. A positive correlation was observed between NO2 −/NO3 − concentrations and JADAS-27 and ESR (R=0.542, p=0.039; R=0.621, p=0.009). Determination of TBARs There were no statistically significant differences in TBAR levels between children with JIA and the control group (0.59±0.2 and 0.61±0.18 μmol/L, respectively) (Table 2). No statistically significant difference was observed in se- rum TBAR levels between children with polyarthritis and oligoarthritis (p>0.05). Determination of TBARs Figure 1 shows higher serum concentrations of NO end-prod- ucts and mean obtained from children with JIA compared to 20 healthy children. Measurement of lipid peroxidation products was based on thiobarbituric acid-reactive substance (TBAR) concentration in plasma. Briefly, 1 mL of 0.05 mol/L H2SO4 and 0.5 mL of 1.23 mol/L trichloroacetic acid (TCA) were added to 0.02 mL of plasma, mixed, and then centrifuged for 10 min (1,500×g, 20 °C). Fluorescence was measured at an excitation wave- length of λ=515 nm and emission wavelength of λ=546 nm. Total NO2 −/NO3 −concentrations in the serum samples of 34 children with JIA were significantly higher (9.59± 4.14μmol/L) than those measured in sera of 20 sex- and age-matched children from the control group (5.59± 2.25 μmol/L; p=0.00069). There was no statistically signifi- cant difference in serum NO2 −/NO3 −concentrations between children with polyarthritis and oligoarthritis (p>0.05) (Table 2). NO assay The concentration of NO was assessed with an indirect method by measurement of NO end-products nitrite/ nitrate (NO2 −/NO3 −) (Nitrate/Nitrite Colorimetric Assay Kit, Sigma-Aldrich Chemie GmbH, Switzerland). Total Twenty sex- and age-matched healthy children (without autoimmune diseases) from the control group were also examined. 453 Clin Rheumatol (2015) 34:451–456 NO2 −/NO3 −concentrations in the serum samples were de- termined by microcolorimetric Griess reaction for nitrite [12] following nitrate reductase-mediated nitrate ion reduction [13]. The absorbance of the chromophore was read at a wavelength of λ=548 nm. Results The demographic, clinical, and laboratory features of children with JIA are shown in Table 1. Statistical analysis Data was expressed as mean and standard deviation (±SD). Results were analyzed using nonparametric Mann-Whitney tests, and correlation was found by the Spearman coefficient. Confidence level was 95 % and statistical significance was considered at a level of 0.05. TBARs did not correlate with either clinical or laboratory variables of disease activity. children with JIA control group 0 2 4 6 8 10 12 14 16 18 20 22 NO3+NO2 [µmol/L] Fig. 1 Nitrite/nitrate (NO2 −/NO3 −) serum concentration and mean value (horizontal line) in children with JIA and in the control group control group children with JIA Clin Rheumatol (2015) 34:451–456 454 FRAP assay Table 2 Concentration of nitrite/nitrate (NO2 −/NO3 −), thiobarbituric acid-reactive substance (TBARs), and the ferric reducing ability of plas- ma (FRAP) in sera of children with JIA and the control group Children with JIA (n=34) Children from control group (n=20) p value NO2 −/NO3 −(μmol/L) 9.59±4.14 5.59±2.25 0.00069 TBARs (μmol/L) 0.59±0.2 0.61±0.18 0.836 FRAP (mmol/L) 0.75±0.1 0.89±0.17 0.048 Table 2 Concentration of nitrite/nitrate (NO2 −/NO3 −), thiobarbituric acid-reactive substance (TBARs), and the ferric reducing ability of plas- ma (FRAP) in sera of children with JIA and the control group evidence for increased endogenous NO synthesis, suggesting that overproduction of NO in an inflamed joint may be im- portant for the pathogenesis of RA, but in JIA its role is still unclear [5–9, 16]. To the best of our knowledge, this is the first study assessing the NO end-products nitrite/nitrate, the index of lipid peroxidation (TBARs), and the total antioxidant plas- ma capacity (FRAP) in children with JIA. The currently reported data with adult RA patients and confirmed in animal models indicated that NO may func- tion as a disease marker and as a proinflammatory medi- ator in arthritis [6–9]. Regarding the pathogenesis of RA, it seems that excessive oxidative stress in the inflamed joints is reflected as an increased concentration of NO in peripheral blood [16]. Our study demonstrated a statisti- cally significant increase in serum concentration of NO2 −/ NO3 −in children with JIA compared to healthy subjects, which is in concordance with results obtained by Lotito et al. and Beri et al. [17, 18]. FRAP assay In the group of children with JIA, FRAP was lower (0.75± 0.1 mmol/L) than in healthy subjects (0.89±0.17 mmol/L, p=0.048) (Fig. 2). However, no statistically significant differ- ence was observed in serum FRAP levels between children with polyarthritis and oligoarthritis (p>0.05). Similarly to other studies, there was no significant differ- ence in NO levels among two JIA subtypes (poly- and oligoarthritis) in our study group [18–20]. Furthermore, statistically significant negative correlations between WBC vs. FRAP levels (R=−0.583, p=0.013) as well as CRP vs. FRAP levels (R=−0.633, p=0.0064) in the group of children with JIA were detected. Similarly to Bica et al. and Lotito et al., we showed that the concentration of NO end-products in sera of children with JIA and active disease was higher than that in children with inactive process, however without a statistically significant difference [3, 17]. Our study indicated a significant positive correlation between JADAS-27 and ESR and NO end- products’ levels, which is in concordance with previous au- thors’ observations in adult patients with RA; however, in JIA such correlations were not found [3, 9, 18]. On the other hand, it was observed by Bica et al. that patients with active JIA and erosive disease had significantly higher NO levels, whereas No statistically significant correlations were detected be- tween distribution of NO end-products, TBARs, and FRAP according to sex, age, and treatment. References 1. Cassidy JT (2001) Juvenile idiopathic arthritis. In: Cassidy JT, Petty RE (eds) Textbook of pediatric rheumatology. Saunders, Philadelphia, pp 218–221 2. Prakken B, Albani S, Martini A (2011) Juvenile idiopathic arthritis. Lancet 377(9783):2138–2149 3. Bica BE, Gomes NM, Fernandea PD, Luiz RR, Koatz VL (2007) Nitric oxide levels and the severity of juvenile idiopathic arthritis. Rheumatol Int 27(9):819–825 4. Labunskyy VM, Gladyshev VN (2012) Role of reactive oxygen species-mediated signaling in aging. Antioxid Redox Signal 19(12): 1362–1372 It is worth noticing that this is the first study on FRAP in children with JIA. Among various methods, assessing the antioxidant capacity of plasma FRAP, which estimates the total antioxidant plasma capacity, seems to be the best, as it gathers whole scavenging abilities of plasma. It is still undecided if a lower level of FRAP is the cause or effect of rheumatoid process. Sarban et al. have described decreased levels of the antioxidant plasma capacity assayed in terms of FRAP in RA patients and postulated that decreased FRAP could contribute to the pathogenesis of rheumatoid process [23]. Although there was no statistically significant difference in FRAP levels, com- parison of our study and control groups indicated that FRAP was lower in sera of children with JIA. Perhaps, a small difference between FRAP levels in the study and control groups was due to the efficient defense against ROS in young patients with JIA or an effective anti-inflammatory therapy of JIA that results in adaptive increase of FRAP [25]. Although there was no significant correlation of FRAP with disease activity, it should be underlined that FRAP negatively corre- lates with CRP and WBC in children with JIA. 5. Chiavaroli V, Giannini C, De Marco S, Chiarelli F, Mohn A (2011) Unbalanced oxidant-antioxidant status and its effects in pediatric diseases. Redox Rep 16(3):101–107 6. Wruck CJ, Fragoulis A, Gurzynski A, Brandenburg LO, Kan YW, Chan K et al (2011) Role of oxidative stress in rheumatoid arthritis: insights from the Nrf2-knockout mice. Ann Rheum Dis 70(5):844– 850 7. Stamp LK, Khalilova I, Tarr JM, Senthilmohan R, Turner R, Haigh RC et al (2012) Myeloperoxidase and oxidative stress in rheumatoid arthritis. Rheumatology (Oxford) 51(10):1796–1803 8. Seven A, Güzel S, Aslan M, Hamuryudan V (2008) Lipid, protein, DNA oxidation and antioxidant status in rheumatoid arthritis. Clin Biochem 41(7–8):538–543 9. Discussion Proinflammatory factors such as cytokines and prostaglandins are released at inflammation sites together with ROS [15]. Several studies in patients with RA have documented children with JIA control group 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 FRAP [mmol/L] Fig. 2 FRAP serum concentration and mean value (horizontal line) in children with JIA and in the control group Fig. 2 FRAP serum concentration and mean value (horizontal line) in children with JIA and in the control group Fig. 2 FRAP serum concentration and mean value (horizontal line) in children with JIA and in the control group FRAP children with JIA 455 Clin Rheumatol (2015) 34:451–456 children with an inactive rheumatoid process had similar NO levels regardless of whether erosions were present or not [3]. children with an inactive rheumatoid process had similar NO levels regardless of whether erosions were present or not [3]. Conflict of interest The authors declare that they have no competing interests. Open AccessThis article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited. Conclusions levels regardless of whether erosions were present or not [3]. There is no compliance among authors about the serum concentration of lipid peroxidation products measured as TBARs in children with JIA. However, it is well established that overproduction of ROS in pathological processes, like chronic inflammation, could result in oxygenation of cell membrane compounds (protein structure damage, lipid oxida- tion), thus inducing loss of cell integrity and functional alter- ation of cell receptors and enzymes [15]. In our group of children with JIA, no statistically significant difference was detected in the concentration of TBARs either between the two JIA subtypes and healthy subjects, which is in line with previous observations of other authors [20]. In contrary, in another study, increased TBARs were found in sera of chil- dren with JIA [21]. Ramos et al. also observed such correla- tions; however, the authors determined different products of lipid peroxidation than we did [19]. Guney et al. who deter- mined malonodialdehyde (MDA) by thiobarbituric acid test indicated higher MDA levels in patients with JIA, with the highest concentration in systemic JIA [15]. The discrepancies between studies may be also explained by the fact that TBARs are not a very specific marker, and perhaps it would be better to determine specific substances like MDA and lipoperoxide (LPO). Furthermore, patients with rheumatoid process have defective defense mechanisms against ROS [6–9, 22, 23]. It was also proved that inflammatory immune response is upon aging [4, 24]. Perhaps, it could be speculated that similar levels of TBARs in healthy children and those with JIA could be explained by high efficiency of the antioxidant system in children, as well as higher cell membrane stability in young subjects and less susceptibility of the membrane’s components to damaging factors [15]. The results provide some evidence for a potential role of the increased NO and decreased antioxidant plasma capacity in the pathogenetic mechanism of JIA. Measurement of NO and FRAP may provide a useful tool to aid in the assessment of the patient’s oxidative stress status and in the determination of an appropriate treatment management plan. Further studies are needed to determine the exact role of ROS and particularly NO in the pathogenesis of JIA and to show its usefulness as a marker of disease activity. Acknowledgments This study is supported by Grants 502-03/8-000- 01/502-64-030 and 503/0-079-05/503/1 from the Medical University of Lodz, Poland. Conclusions Conflict of interest The authors declare that they have no competing interests. References Nagy G, Koncz A, Telarico T, Fernandez D, Ersek B, Buzás E et al (2010) Central role of nitric oxide in the pathogenesis of rheumatoid arthritis and systemic lupus erythematosus. Arthritis Res Ther 12(3): 210 10. Petty RE, Southwood TR, Manners P, Baum J, Glass DN, Goldenberg J et al (2004) International League of Associations for Rheumatology classification of juvenile idiopathic arthritis; second revision, Edmonton, 2001. J Rheumatol 31:390–392 11. Consolaro A, Ruperto N, Bazso A, Pistorio A, Magni-Manzoni S, Filocamo G et al (2009) Paediatric Rheumatology International Trials 456 Clin Rheumatol (2015) 34:451–456 Organisation: Development and validation of a composite disease activity score for juvenile idiopathic arthritis. Arthritis Rheum 61(5): 658–666 19. Ramos VA, Ramos PA, Dominguez MC (2000) The role of oxidative stress in inflammation in patients with juvenile rheumatoid arthritis. J Peditr (Rio J) 76(2):125–132 12. Ding AH, Nathan CF, Stuehr DJ (1988) Release of reactive nitrogen intermediates and reactive oxygen intermediates from mouse peritoneal macrophages. Comparison of activating cyto- kines and evidence for independent production. J Immunol 141(7):2407–2412 20. Gromadzinska J, Sklodowska M, Wolkanin P, Biernacka M, Brozik H, Wasowicz W et al (1993) The activity of some trace element- dependent enzymes in blood of children with juvenile chronic arthri- tis. TEMA 8:780–783 21. Sklodowska M, Gromadzińska J, Biernacka M, Wasowicz W, Wolkanin P, Marszalek A et al (1996) Vitamin E, thiobarbituric acid reactive substance concentrations and superoxide dismutase activity in the blood of children with juvenile rheumatoid arthritis. Clin Exp Rheumatol 14(4):433–439 13. Gilliam MB, Sherman MP, Griscavage JM, Ignarro LJ (1993) A spectrophotometric assay for nitrate using NADPH oxida- tion by Aspergillus nitrate reductase. Anal Biochem 212(2): 359–365 14. Benzie IFF, Strain JJ (1996) The ferric reducing ability of plasma (FRAP) as a measure of “antioxidant power”. The FRAP assay. Anal Biochem 239:70–76 22. Hirao M, Yamasaki N, Oze H, Ebina K, Nampei A, Kawato Y et al (2012) Serum level of oxidative stress marker is dramatically low in patients with rheumatoid arthritis treated with tocilizumab. Rheumatol Int 32(12):4041–4045 15. Guney T, Yildiz B, Altikat S, Kural N, Alatas O (2009) Decreased antioxidant capacity and increased oxidative stress in patients with juvenile idiopathic arthritis. JPS 1(e3):1–6 23. Sarban S, Kocyigit A, Yazar M, Isikan UE (2005) Plasma total antioxidant capacity, lipid peroxidation, and erythrocyte antioxidant enzyme activities in patients with rheumatoid arthritis and osteoar- thritis. Clin Biochem 38:981–986 16. References Brik R, Rosen I, Savulescu D, Borovoi I, Gavish M, Nagler R (2010) Salivary antioxidants and metalloproteinases in juvenile idiopathic arthritis. Mol Med 16(3–4):122–128 24. Lee K, Won HY, Bae MA, Hong JH, Hwang ES (2011) Spontaneous and aging-dependent development of arthritis in NADPH oxidase 2 deficiency through altered differentiation of CD11b + and Th/Treg cells. Proc Natl Acad Sci U S A 108(23):9548–9553 17. Lotito AP, Muscara MN, Kiss MH, Teixeira SA, Novaes GS, Laurindo IM et al (2004) Nitric oxide-derived species in synovial fluid from patients with juvenile idiopathic arthritis. J Rheumatol 31(5):992–997 25. Renke J, Szlagatys A, Hansdorfer-Korzon R, Szumera M, Kamińska B, Knap N et al (2007) Persistence of protein oxidation products and plasma antioxidants in juvenile idiopathic arthritis. A one-year follow-up study. Clin Exp Rheumatol 25(1):112–114 18. 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https://openalex.org/W1986805956	https://sajhrm.co.za/index.php/sajhrm/article/download/464/627	English	null	Confirming theoretical pay constructs of a variable pay scheme	SA journal of human resource management	2,013	cc-by	1,352	Appendix 1 TABLE 1: Relationship between items, dimensions and constructs. Construct Dimension Cronbach’s alpha Item number 1. Performing (0.872) 1.1. Performance culture 0.806 1–5 1.2. Performance management 0.794 6–11 2. Instrumentality (0.799) 2.1. Certainty 0.649 12–16 2.2. Exclusivity 0.505 17–19 2.3. Trust 0.702 20–24 2.4. Risk 0.465 25–27 3. Congruency (0.971) 3.1. External alignment 0.913 28–39 3.2. Internal alignment 0.902 40–50 3.3. Understanding and acceptance 0.929 51–67 3.4. Continuous improvement 0.820 68–71 3.5. Goal difficulty 0.817 72–78 Source: Data collected by authors during present study TABLE 1: Relationship between items, dimensions and constructs. Construct Dimension TABLE 1: Relationship between items, dimensions and constructs. Source: Data collected by authors during present study TABLE 2: Structure equation modelling results. TABLE 2: Structure equation modelling results. Page 1 of 2 Page 1 of 2 Appendix 1 Model Mardia’s coefficient df χ2 χ2/df S-Bχ2 value S-Bχ2/df CFI SRMR RMSEA RMSEA 90% CI Performance culture (all items) 15.72 5 11.39 2.28 9.23 1.85 ML (0.985) ML (0.028) ML (0.066) 0.012: 0.117 RML (0.985) – RML (0.054) 0.000: 0.107 Performance management (all items) 16.90 9 63.53 7.06 36.30 4.03 ML (0.938) ML (0.047) ML (0.145) 0.113: 0.178 RML (0.963) – RML (0.103) 0.070: 0.138 Certainty (all items) 5.52 5 6.89 1.38 5.26 1.05 ML (0.993) ML (0.029) ML (0.040) 0.000: 0.102 RML (0.999) – RML (0.017) 0.000: 0.090 Certainty (omitted A14) 5.08 2 0.03 0.01 0.02 0.01 ML (1.000) ML (0.003) ML (0.000) – RML (1.000) – RML (0.000) – Exclusivity (all items) 5.67 5 18.53 3.71 12.47 2.49 ML (0.926) ML (0.056) ML (0.106) 0.058: 0.157 RML (0.949) – RML (0.079) 0.027: 0.133 Exclusivity (omitted A19 B) 4.42 2 0.42 0.21 0.42 0.21 ML (1.000) ML (0.009) ML (0.000) 0.000: 0.079 RML (1.000) – RML (0.000) 0.000: 0.068 Trust (all items) 4.50 5 19.87 3.97 19.87 3.97 ML (0.939) ML (0.055) ML (0.111) 0.064: 0.162 RML (0.939) – RML (0.096) 0.048: 0.148 Trust (omitted A22 and A24) 4.30 – -0.03 – -0.03 – – – – – Risk (all items) 3.97 – -0.03 – -0.03 – – – – – External alignment (all items) 34.14 54 255.85 4.74 154.13 2.85 ML (0.837) ML (0.071) ML (0.129) 0.114: 0.145 RML (0.891) RML (0.092) 0.075: 0.108 Internal alignment (all items) 39.23 44 182.95 4.16 96.88 2.20 ML (0.904) ML (0.051) ML (0.122) 0.105: 0.139 RML (0.953) – RML (0.077) 0.058: 0.095 Understanding and acceptance (all items) recoded A66 46.62 119 549.96 4.62 294.86 2.48 ML (0.834) ML (0.064) ML (0.127) 0.116: 0.137 RML (0.913) – RML (0.081) 0.070: 0.092 Understanding and acceptance (omitted rA66 and A67) 48.54 90 459.52 5.11 230.20 2.56 ML (0.850) ML (0.059) ML (0.135) 0.123: 0.147 RML (0.928) – RML (0.084) 0.071: 0.096 Continuous improvement (all items) 13.96 2 8.69 4.34 4.08 2.04 ML (0.977) ML (0.033) ML (0.126) 0.053: 0.212 RML (0.989) – RML (0.072) 0.000: 0.165 Goal efficacy alignment (all items) 17.80 14 101.62 7.26 64.80 4.63 ML (0.833) ML (0.106) ML (0.168) 0.138: 0.198 RML (0.864) – RML (0.128) 0.098: 0.159 Goal efficacy alignment (omitted A72, A73 and A74) 13.46 2 2.68 1.34 1.33 0.66 ML (0.998) ML (0.014) ML (0.041) 0.000: 0.143 RML (1.000) – RML (0.000) 0.000: 0.116 Performing (all items) 26.62 43 185.92 4.32 129.84 3.02 ML (0.903) ML (0.078) ML (0.107) 0.092: 0.123 RML (0.923) – RML (0.084) 0.068: 0.100 Instrumentality (all items) 25.86 129 536.82 4.16 404.76 3.14 ML (0.730) ML (0.142) ML (0.113) 0.104: 0.123 RML (0.777) – RML (0.094) 0.083: 0.103 Instrumentality (omitted A14, A19b, A22 and A24) 23.62 71 149.75 2.11 106.40 1.50 ML (0.928) ML (0.052) ML (0.069) 0.055: 0.084 RML (0.958) – RML (0.048) 0.030: 0.064 Congruency (all items) 77.07 1214 3265.60 2.69 2076.53 1.71 ML (0.740) ML (0.085) ML (0.088) 0.084: 0.091 RML (0.853) – RML (0.058) 0.054: 0.061 Source: Data collected by authors during present study df, degrees of freedom; χ2, Chi-squared; S-Bχ2, normed Chi-squared; CFI, comparative fit index; SRMR, standardised root mean square residual; RMSEA, root mean square error of approximation; CI, confidence index. Note: This is the Online Appendix of Ncube, S., Bussin, M.H.R., & De Swardt, L. (2013). Confirming theoretical pay constructs of a variable pay scheme. SA Journal of Human Resource Management/SA Tydskrif vir Menslikehulpbronbestuur, 11(1), Art. #464, 10 pages. http://dx.doi.org/10.4102/sajhrm.v11i1.464 Appendix 1 http://www.sajhrm.co.za doi:10.4102/sajhrm.v11i1.464-1 TABLE 2 (Continues...): Structure equation modelling results. Model Mardia’s coefficient df χ2 χ2/df S-Bχ2 value S-Bχ2/df CFI SRMR RMSEA RMSEA 90% CI Congruency (omitted rA66, A67, A72, A73 and A74) 77.95 979 2687.62 2.75 1664.75 1.70 ML (0.771) ML (0.071) ML (0.089) 0.085: 0.093 RML (0.878) – RML (0.057) 0.053: 0.061 Congruency external and internal alignment together (highly correlated) 77.95 983 2777.02 2.83 1720.92 1.75 ML (0.760) ML (0.071) ML (0.091) 0.087: 0.095 RML (0.869) – RML (0.059) 0.055: 0.063 Congruency external and internal alignment together (omitted A28-A31, A36 and A37) 72.20 734 2115.32 2.88 1297.41 1.77 ML (0.789) ML (0.067) ML (0.093) 0.088: 0.097 RML (0.888) – RML (0.060) 0.055: 0.065 Alignment (all Items) 65.14 230 863.12 3.75 490.98 2.13 ML (0.804) ML (0.067) ML (0.111) 0.103: 0.119 RML (0.893) – RML (0.072) 0.063: 0.080 Alignment (omitted A28-A31, A36 and A37) 53.07 119 435.15 3.66 239.69 2.01 ML (0.864) ML (0.057) ML (0.111) 0.100: 0.121 RML (0.932) – RML (0.070) 0.058: 0.081 Source: Data collected by authors during present study df, degrees of freedom; χ2, Chi-squared; S-Bχ2, normed Chi-squared; CFI, comparative fit index; SRMR, standardised root mean square residual; RMSEA, root mean square error of approximation; CI, confidence index. TABLE 3: Synergies between research on variable pay plans. Revised variable remuneration model (2012) Mathis and Jackson’s (2008) findings Greene’s (2011) findings Organisational fit Align behaviour outcome to incentive plan Congruency Alignment Consistency with organisational objectives - Plan objective Efficacy/goal difficulty - - - Understanding Clear communication of plan - - Transparency Updated on the detail of the performance achieve - - Continuous improvement - - - Instrumentality Trust Clear differentiation with base pay - Payment timing Certainty - - Administration Exclusivity - - Eligibility Business continuity - - - Performing Performance management - Link performance results with meaningful reward - - Fair performance measures - - Plan performance link to desirable behaviour - Performance culture - Availability of sufficient financial resources (This factor was grouped under the cluster ‘Organisational fit’) - Participant involvement Building the pool - - Formula funding Distribution of incentive - - Formula distribution - - Plan types Sources: For more information, please see the full reference list of the article, Ncube, S., Bussin, M.H.R., & De Swardt, L. (2013). Confirming theoretical pay constructs of a variable pay scheme. SA Journal of Human Resource Management/SA Tydskrif vir Menslikehulpbronbestuur, 11(1), Art. #464, 10 pages. Appendix 1 http://dx.doi.org/10.4102/sajhrm.v11i1.464 TABLE 3: Synergies between research on variable pay plans. Revised variable remuneration model (2012) Mathis and Jackson’s (2008) findings Greene’s (2011) findings Organisational fit Align behaviour outcome to incentive plan Congruency Alignment Consistency with organisational objectives - Plan objective Efficacy/goal difficulty - - - Understanding Clear communication of plan - - Transparency Updated on the detail of the performance achieve - - Continuous improvement - - - Instrumentality Trust Clear differentiation with base pay - Payment timing Certainty - - Administration Exclusivity - - Eligibility Business continuity - - - Performing Performance management - Link performance results with meaningful reward - - Fair performance measures - - Plan performance link to desirable behaviour - Performance culture - Availability of sufficient financial resources (This factor was grouped under the cluster ‘Organisational fit’) - Participant involvement Building the pool - - Formula funding Distribution of incentive - - Formula distribution - - Plan types Sources: For more information, please see the full reference list of the article, Ncube, S., Bussin, M.H.R., & De Swardt, L. (2013). Confirming theoretical pay constructs of a variable pay scheme. SA Journal of Human Resource Management/SA Tydskrif vir Menslikehulpbronbestuur, 11(1), Art. #464, 10 pages. http://dx.doi.org/10.4102/sajhrm.v11i1.464 Sources: For more information, please see the full reference list of the article, Ncube, S., Bussin, M.H.R., & De Swardt, L. (2013). Confirming theoretical pay Journal of Human Resource Management/SA Tydskrif vir Menslikehulpbronbestuur, 11(1), Art. #464, 10 pages. http://dx.doi.org/10.4102/sajhrm.v11i1.46 doi:10.4102/sajhrm.v11i1.464-1 http://www.sajhrm.co.za http://www.sajhrm.co.za doi:10.4102/sajhrm.v11i1.464-1
https://openalex.org/W3140457102	https://www.seer.ufal.br/index.php/revistaleitura/article/download/10501/7906	Portuguese	null	Uma análise do leitor presumido em rótulos de embalagens de produtos capilares	Leitura	2,020	cc-by	7,699	Resumo Ao se comunicar, o usuário da língua constrói o seu dizer guiado por um objetivo e visando o seu interlocutor. Assim, o leitor presumido é um fenômeno considerado como condição necessária para que um texto seja desenvolvido. Baseado nos estudos de base sociorretórica, objetivamos analisar a construção do leitor presumido em rótulos de embalagens de produtos capilares. Para tanto, nos apoiaremos em Bazerman (2006), Miller (2009 [1984]), Koch (2016), Bakhtin (2003 [1979]; 2006) e Souza (2008). Esta pesquisa possui um corpus composto por dois rótulos de embalagens de produtos para cabelos cacheados e, para uma melhor compreensão do contexto histórico, cultural e social desse gênero, apoia-se ainda em alguns dados do Dossiê BrandLab: A Revolução dos Cachos e do Caderno de tendências 2019–2020 da ABIHPEC e SEBRAE, coletados na internet. As análises revelam que o leitor presumido dos rótulos de embalagens de produtos para cabelos cacheados analisados nesta pesquisa, em sua maioria, faz parte de um público feminino jovem que está conectado à internet e às redes sociais e é tratado com intimidade pelo produtor do texto. Concluímos que esse gênero se configura como registros históricos de valores culturais e ideológicos da contemporaneidade, perceptíveis na dinâmica locutor/interlocutor. Palavras-chave: Estudos Retóricos de Gêneros. Leitor Presumido. Gênero rótulos de embalagens An analysis of the presumed reader on packaging labels of hair products Felipe Augusto de Sousa Sobrinho 1 Maria Eliana Ferreira Fernandes 2 Felipe Augusto de Sousa Sobrinho 1 Maria Eliana Ferreira Fernandes 2 Leitura, Maceió, n. 66, set./dez. 2020 – ISSN 2317-9945. Estudos linguísticos e literários, p. 130-145. 1 Mestre em Letras pela Universidade Estadual do Piauí (2020). Professor de Língua Portuguesa vinculado à Secretaria de Educação do Estado do Ceará. ç 2 . Mestre em Letras pelo Programa de Pós- Graduação em Letras da Universidade Estadual do Piauí. Introdução Os estudiosos da sociorretórica entendem que os gêneros são parte integrante da sociedade (BAZERMAN, 2006; MILLER, 2009 [1984]) e consideram que o estudo dos gêneros é mais sólido quando considera o texto-em-contexto, o seu entorno social. Nossas ações linguísticas se apoiam em algum gênero textual. Quando produzimos um texto de determinado gênero, somos guiados por um objetivo e visamos um leitor presumido, o qual recebe diferentes denominações tanto pela Linguística Textual (doravante LT) quanto pela teoria de gêneros. Nosso objetivo é identificar, descrever e analisar o leitor presumido do gênero rótulo de embalagens de produtos a partir de suas pistas linguísticas e contextuais. Para tanto, procuraremos neste estudo, primeiramente, apresentar algumas concepções de gênero na perspectiva bakhtiniana e na sociorretórica. Em seguida, observaremos algumas denominações sobre leitor presumido feitas pela LT e pelas teorias de gêneros; e apresentaremos ainda algumas características e funcionalidades do gênero rótulo de embalagens. E, por fim, faremos a apresentação de algumas considerações a partir dos resultados da análise de dois rótulos de embalagens dos produtos para cabelos cacheados. Abstract When communicating, the language user constructs his speech guided by a purpose and aiming at his interlocutor. Thus, the presumed reader is a phenomenon considered as a necessary condition for a text to be developed. Based on socio-rhetorical studies, we aim to analyze the construction of the presumed reader on packaging labels of hair products. For that, we will support on Bazerman (2006), Miller (2009 [1984]), Koch (2016), Bakhtin (2003 [1979]; 2006) and Souza (2008). This research has a corpus composed of two packaging labels of products for curly hair and, for a better understanding of the historical, cultural and social context of this genre, it is also supported by some data from the BrandLab Dossier: The Revolution of curls and from the trends notebook 2019-2020 from ABIHPEC and SEBRAE, collected on the internet. The analyzes reveal that the presumed reader of the packaging labels of products for curly hair produced in the contemporary context, mostly, is part of a young female audience that is connected to the internet and social networks and is treated with intimacy by the producer of the text. We conclude that this genre is configured as historical records of contemporary cultural and ideological values, noticeable in the speaker / interlocutor dynamics. Keywords: Rhetorical Gender Studies. Presumed Reader. Genre packaging labels Recebido em: 01/07/2020. Aceito em: 20/10/2020. 1 Mestre em Letras pela Universidade Estadual do Piauí (2020). Professor de Língua Portuguesa vinculado à Secretaria de Educação do Estado do Ceará. 2 . Mestre em Letras pelo Programa de Pós- Graduação em Letras da Universidade Estadual do Piauí. 130 130 Leitura, Maceió, n. 66, set./dez. 2020 – ISSN 2317-9945. Estudos linguísticos e literários, p. 130-145. O conceito de gênero na perspectiva bakhtiniana e nos estudos sociorretóricos de gênero O caráter sociointeracional de abordagem dos gêneros iniciou-se com Bakhtin (2003 [1979]), teórico que define os gêneros do discurso como “tipos relativamente estáveis de enunciado” (p. 279), visto que eles podem sofrer adaptações em sua forma e função, pois acompanha a evolução social e as necessidades de comunicação da sociedade. Na perspectiva bakhtiniana, os gêneros podem se transformar e se adaptar, conforme o contexto de produção, circulação e recepção exigirem. Bakhtin (2003, p. 262) entende que os gêneros acompanham a dinamicidade social e pontua que há riqueza e diversidade de gêneros discursivos porque “são inesgotáveis as possibilidades da multiforme atividade humana e porque em cada campo dessa atividade é integral o repertório de gêneros de discurso, que cresce e se diferencia à medida que se desenvolve e se complexifica um determinado campo”. Por sua constituição ser determinada historicamente, Bakhtin (2003) defende que os gêneros são investidos de ideologias relacionadas a interesses de classes, pois, em sua visão dialógica, o sujeito- enunciador se constitui pela linguagem na relação entre o eu (sujeito) e o outro (contexto social). Nesse sentido, o pensamento bakhtiniano destaca que as condições exteriores e o contexto social imediato determinam toda enunciação. De acordo com Bakhtin (2006), o enunciado se dirige a alguém e se concretiza em função dessa orientação, pois: [...] a personalidade que se exprime, apreendida, por assim dizer, do interior, revela-se um produto total da inter-relação social. A atividade mental do sujeito constitui, da mesma forma que a expressão exterior, um território social. Em consequência, todo o itinerário que leva da atividade mental (o “conteúdo a exprimir”) à sua objetivação externa (a “enunciação”) situa-se completamente em território social (BAKHTIN, 2006, p. 121-122). Leitura, Maceió, n. 66, set./dez. 2020 – ISSN 2317-9945. Estudos linguísticos e literários, p. 130-145. 131 131 131 Desse postulado, entende-se que quando o sujeito se propõe a comunicar algo, ele elabora uma proposta comunicativa, situando-se em seu território social, e com base nessa proposta e nesse território ele organiza seu pensamento. Nesse momento, conforme a teoria bakhtiniana, a escolha de um gênero que atenda ao seu propósito de dizer e a elaboração do enunciado e de seu meio de divulgação também entram em jogo, tendo em vista o leitor visado. Já nos estudos sociorretóricos de gênero, a noção de gênero como ação social prevalece. O conceito de gênero na perspectiva bakhtiniana e nos estudos sociorretóricos de gênero Compreender gêneros na abordagem sociorretórica (MILLER, 2009 [1984]; BAZERMAN, 2006) implica concebê-los como ações sociais tipificadas que estão diretamente relacionadas com as situações retóricas, as quais também são tipificadas. Dentro dessa concepção, admite-se que os gêneros são estruturas semióticas dinâmicas, pois estão constantemente mudando e se ajustando às práticas sociais de modo complexo e dinâmico. Na perspectiva sociorretórica, incorporam-se interesses e valores de um grupo social em particular e reforçam-se regras sociais e relações entre escritores e leitores. Miller (2009 [1984], p. 24) afirma que os gêneros são uma “forma de ação social”, que envolvem situação e motivo. Conforme essa pesquisadora, a ação humana “só pode ser interpretada dentro de um contexto situacional e por meio de atribuição de motivos”. Essa abordagem traz o entendimento de que os textos têm efeitos sobre a vida das pessoas. Dessa maneira, para a compreensão dos significados é preciso entender também a relação entre os participantes do evento social em que o texto se insere. Bazerman (2006, p.23) corrobora essa ideia, ao afirmar que os gêneros “são formas de vida, modos de ser. São frames para a ação social. São ambientes para a aprendizagem. São os lugares onde o sentido é construído. Os gêneros moldam os pensamentos que formamos e as comunicações através das quais interagimos”. Para esse autor, os usuários da língua organizam suas ações diárias pelo uso de textos e dão significado a um processo interativo tipificado num sistema de atividades que encadeia significativamente as ações discursivas. Nesse sentido, Bazerman (2006) compreende que os gêneros surgem de um viés histórico, cultural e interativo dentro de instituições e atividades anteriormente já existentes. Alves Filho e Soares de Sá (2015) afirmam que o caráter social de ações desenvolvidas em torno da linguagem permite que o uso dos gêneros não seja confuso, pois essas ações sociais tanto servem para provocar alterações como para manter a estabilidade dos gêneros; já que os indivíduos sozinhos não conseguem mudar a forma e a função dos gêneros, tudo é feito social e coletivamente. Acreditamos que no fenômeno escolhido como objeto de estudo deste trabalho, é possível perceber a relatividade do gênero e o seu atendimento à necessidade humana. Assim, por considerarem a função social dos gêneros, as definições bakhtinianas de gêneros e as da abordagem sociorretórica servem para subsidiar a abordagem do leitor presumido e do gênero rótulo de embalagens de produtos capilares que apresentamos a seguir. Leitura, Maceió, n. 66, set./dez. 2020 – ISSN 2317-9945. Estudos linguísticos e literários, p. 130-145. O leitor presumido na linguística textual e na teoria dos gêneros A figura do leitor presumido constitui-se condição necessária para que um texto seja desenvolvido. Afinal, quando produzimos um texto, independente do gênero a que 132 pertence, sempre pressupomos um leitor para ele. Por essa razão, consideramos ser uma importante categoria teórica para ser analisada nos rótulos de embalagens de produtos capilares. O leitor presumido tem recebido diferentes denominações tanto pela Linguística Textual quanto pela Teoria de gêneros. O que a Linguística textual denomina de interlocutor visado ou interactante (KOCH, 2016), os teóricos de gêneros denominam de auditório social (BAKHTIN, 2006), ou ainda audiência (MILLER, 2009 [1984]). Como os textos necessariamente se vinculam aos gêneros, é fundamental analisarmos o texto considerando a inter-relação existente entre texto, gênero e discurso; por isso, optamos por apresentar noções sobre o leitor presumido feitas tanto pela LT quanto pela teoria de gêneros. Além disso, consideraremos a prática e o contexto social em que o gênero investigado neste estudo é produzido. Koch (2016), ao refletir sobre o texto e a construção dos sentidos dentro de uma perspectiva sociointeracionista de estudo da linguagem, reconhece a influência e a importância dos sujeitos interactantes nas análises textuais. Conforme a autora: As teorias sociointeracionais reconhecem a existência de um sujeito planejador/organizador que, em sua inter-relação com outros sujeitos, vai construir um texto, sob a influência de uma complexa rede de fatores, entre os quais a especificidade da situação, o jogo de imagens recíprocas, as crenças, convicções, atitudes dos interactantes, os conhecimentos (supostamente) partilhados, as expectativas mútuas, as normas e convenções socioculturais (KOCH, 2016, p. 7, grifos nossos). O processo de construção de texto, para Koch (2016), envolve diversos fatores tanto cognitivos quanto discursivos e socioculturais. Sendo assim, o leitor presumido também é construído sob esses fatores. Todos eles influenciam na produção e construção dos sentidos do texto. Desse postulado, fica clara a ideia de que o texto deve ser analisado com base nas práticas sociais e culturais que o envolvem. Já Bakhtin (2006), em seus estudos sobre gêneros do discurso, criou para o leitor presumido a denominação auditório social, e ao discorrer sobre a natureza social e ideológica da enunciação, trata-a como discurso interior ou exterior que “não existe fora de um contexto social, já que cada locutor tem um ‘horizonte social’. Há sempre um interlocutor, ao menos potencial. O locutor pensa e se exprime para um auditório social bem definido” (p. 17, grifos nossos). Leitura, Maceió, n. 66, set./dez. 2020 – ISSN 2317-9945. Estudos linguísticos e literários, p. 130-145. Leitura, Maceió, n. 66, set./dez. 2020 – ISSN 2317-9945. Estudos linguísticos e literários, p. 130-145. O leitor presumido na linguística textual e na teoria dos gêneros Para esse teórico, o auditório social equivale à atmosfera em que são construídas as motivações, apreciações e deduções do falante; assim, na visão de Bakhtin, o auditório social constitui-se condição necessária para que um texto seja desenvolvido e é organizado de certa maneira, pois conforme ele afirma, Toda situação inscrita duravelmente nos costumes possui um auditório organizado de uma certa maneira e consequentemente um certo repertório de pequenas fórmulas correntes. A fórmula estereotipada adapta-se, em qualquer lugar, ao canal de interação social que lhe é reservado, refletindo ideologicamente o tipo, a estrutura, os objetivos e a composição social do grupo. As fórmulas da vida corrente fazem parte do meio social, são elementos da festa, dos lazeres, das relações que se travam no hotel, nas fábricas, etc. Elas coincidem com esse meio, são por ele delimitadas e determinadas em todos os aspectos (BAKHTIN, 2006, p. 128). Leitura, Maceió, n. 66, set./dez. 2020 – ISSN 2317-9945. Estudos linguísticos e literários, p. 130-145. 133 133 133 Leitura, Maceió, n. 66, set./dez. 2020 – ISSN 2317-9945. Estudos linguísticos e literários, p. 130-145. Leitura, Maceió, n. 66, set./dez. 2020 – ISSN 2317-9945. Estudos linguísticos e literários, p. 130-145. Na linha dessa discussão de Bakhtin sobre a natureza social e ideológica da enunciação, Pereira e Rodrigues (2014) sublinham nos seus estudos sobre os conceitos de valoração nos estudos do Círculo de Bakhtin, numa relação entre ideologia e linguagem, que um dos objetivos da perspectiva desses estudos se volta para a compreensão de como os discursos são refratados pela ideologia. Para isso, ponderam que os discursos não são neutros, mas carregados por um conceito de valoração, sob um viés de significação social. Como os autores destacam, essa perspectiva de valoração está sustentada na ideologia, que deve ser vista não tendo como foco apenas o psíquico, sobretudo precisa considerar sua materialidade, baseados em Volochinov. Nesse direcionamento, há uma relação entre a ideologia e sua materialidade por meio dos signos. Para discorrer sobre essa visão na prática, Pereira e Rodrigues (2014) sublinham o posicionamento da classe dominante ao apresentar esses signos como não alcançáveis e alheios aos grupos sociais para que os padrões da sociedade não sejam reconhecidos e os signos sejam vistos apenas sob uma validade, ou “monovalência”, termo usado por eles. Os debates sobre as variações linguísticas, por exemplo, retratam essa falsa supremacia. Leitura, Maceió, n. 66, set./dez. 2020 – ISSN 2317-9945. Estudos linguísticos e literários, p. 130-145. O leitor presumido na linguística textual e na teoria dos gêneros É nessa linha que Pereira e Rodrigues (2014) discutem a relação entre ideologia, valoração e linguagem. Para Bakhtin (2006), ideologia é relacionada a diversas formas de cultura, assim como à ideia do psiquismo (consciência individual). Assim, Pereira e Rodrigues (2014, p. 181) afirmam que “os enunciados, que materializam os discursos, apresentam sempre uma dimensão avaliativa e expressam um posicionamento social”, confirmando a visão de Bakhtin, ao assegurar que os enunciados são dirigidos socialmente, sempre levando em conta um interlocutor. Bathkin (2016, p. 60-116) afirma que o enunciado não pode ser considerado de forma isolada, e tendo como foco apenas o seu autor, mas levando em consideração “como um elo na cadeia da comunicação discursiva e da relação com outros enunciados a ele vinculados”. Ainda nesse sentido, o autor ratifica que o enunciado tem um destinatário definido e, assim, considerando esse o “público e suas diferentes épocas”. É nessa visão que focamos o leitor presumido, aquele para o qual o enunciado é dirigido. Nas interações sociais, segundo Bakhtin (2006), produtor e leitor dos textos recorrem a fórmulas correntes e estereotipadas a que tiveram acesso nos grupos sociais em que estão inseridos. Para esse autor, essas fórmulas são determinadas pelo meio social a que seus usuários pertencem. Desse modo, o postulado bakhtiniano reitera a noção de que para se compreender o leitor presumido, ou auditório social, de determinado texto deve-se considerar o seu entorno social. Enquanto na perspectiva bakhtiniana o leitor presumido é tratado como auditório social, nos estudos de Miller (2009 [1984]) auditório social passa a ser denominado “audiência”. Para a autora, a utilização de enunciados presume que o usuário conheça aquele para quem sua construção enunciativa se dirige. Dessa forma, a audiência se refere àqueles que irão antecipar ou responder de certa maneira ao que foi dito (MILLER, 2009 [1984], p. 34). Biasi-Rodrigues (2002), ao comentar a dimensão social que está presente nas noções de gênero mais atuais, como a proposta por Miller (2009 [1984]), que considera os gêneros como forma de ação social, argumenta que: Leitura, Maceió, n. 66, set./dez. 2020 – ISSN 2317-9945. Estudos linguísticos e literários, p. 130-145. 134 134 Leitura, Maceió, n. 66, set./dez. 2020 – ISSN 2317-9945. Estudos linguísticos e literários, p. 130-145. a noção de audiência, ou de uma audiência potencial, sem dúvida, é um fator determinante dessa ação. O leitor presumido na linguística textual e na teoria dos gêneros O sentido de audiência exerce influência direta nas escolhas que o falante/escritor faz, quando em situações de produção: seja do tópico, da quantidade e do balanceamento das informações (mais ou menos explícitas), das estratégias de organização do texto em termos de seleção lexical e de relações semântico-sintáticas, seja do estilo e do registro mais adequados para criar textualidade e interagir com o provável ouvinte/leitor (MILLER, 2009, p. 51). Nessa observação, Biasi-Rodrigues (2002) defende a ideia de que o texto apresenta marcas linguísticas de sua audiência, reforçando, dessa forma, as colocações apresentadas anteriormente de que o texto se organiza visando um leitor pressuposto pelo autor do texto. Segundo Soares de Sá (2013), estudos da socio-historicidade dos gêneros mostram que eles são construtos compartilhados e tipificados. Por essa razão, eles suscitam em seus usuários expectativa de entendimento. O ouvinte/leitor presumido precisa ser pressuposto (mesmo que vagamente) para que a enunciação seja dotada de compreensão, pois, como afirmam Bakhtin/Voloshinov (1976 [1926], p. 117), a palavra “é determinada tanto pelo fato de que procede de alguém como pelo fato de que se dirige para alguém”. Como vemos, essas noções são exatamente as mesmas, mas são denominadas diferentemente por autores de diferentes perspectivas teóricas. Não importa se o pesquisador trata o fenômeno do leitor presumido como interlocutor, auditório social ou audiência, o fundamental é entender que esse fenômeno desempenha papel essencial nas escolhas estilísticas de quem produz um texto, texto este que necessariamente está inserido em determinado gênero. Em suma, o leitor presumido participa de modo ativo na construção do dizer. A seguir, apresentamos algumas das características e funções do gênero que será objeto de estudo neste trabalho: o rótulo de embalagens de produtos capilares. Leitura, Maceió, n. 66, set./dez. 2020 – ISSN 2317-9945. Estudos linguísticos e literários, p. 130-145. O gênero rótulo de embalagens: caraterísticas e funcionalidades Produzidos com o objetivo de informar, identificar e incentivar a comercialização dos produtos postos à venda, os rótulos de embalagens cumprem uma importante função social e se inserem dentro das práticas sociais na esfera de circulação social de produção e consumo. Dentre os inúmeros rótulos de embalagens existentes no mercado, optamos por investigar os de produtos capilares, considerados produtos de higiene pessoal, os quais, para estarem no mercado, são obrigados pela legislação brasileira a ser rotulados, conforme Lei n.º 6.360, de 23 de setembro de 1976. A Agência Nacional de Vigilância Sanitária (ANVISA) estabelece as normas para a constituição dos rótulos e define rotulagem como “toda inscrição, legenda, imagem ou toda matéria descritiva ou gráfica, escrita, impressa, estampada, gravada, gravada em relevo ou litografada ou colada sobre a embalagem” (ANVISA, 2002). A referida Agência estabelece, por meio de um regulamento técnico sobre rotulagem obrigatória geral, as informações necessárias que devem constar nos rótulos de produtos de higiene pessoal relacionadas à utilização e toda a indicação necessária referente ao produto (ANVISA, 2015). Conforme Borges (2016), os textos dos rótulos “servem à identificação do conteúdo dos recipientes e, também, à comercialização de produtos. Ao desempenharem 135 135 Leitura, Maceió, n. 66, set./dez. 2020 – ISSN 2317-9945. Estudos linguísticos e literários, p. 130-145. seu papel, entretanto, eles podem se constituir em registros históricos” (p. 17). Fernandes (2018, p. 97) também entende que o rótulo se caracteriza “como registro histórico, representando a cultura, os valores e os ideais de uma sociedade, numa determinada época, podendo revelar os gostos e as tendências de consumo desta sociedade como também as intenções do produtor”. Para Souza (2008, p. 15), a linguagem dos rótulos pode construir “significados diversos da realidade que os circunda”, revelar o meio social em que eles se inserem e apresentar um aspecto discursivo que permite fazer uma reflexão dos rótulos como um objeto cultural. Conforme a autora, “a criação, a formulação e a composição do gênero fazem parte de uma questão de valoração, pois, quando se produz um rótulo de produto de higiene pessoal, há certas escolhas para informar e “agradar” o consumidor” (SOUZA, 2008, p. 52 [grifos da autora]). Desse modo, esse gênero pode apresentar características particulares a fim de representar uma realidade. O gênero rótulo de embalagens: caraterísticas e funcionalidades Borges (2016) compreende o poder persuasivo do rótulo de embalagens, ao considerá-lo uma espécie de vendedor silencioso que, junto com a embalagem, “pode levar um produto a ser escolhido em um universo de diversos outros, na medida em que o diferencia dos demais e que, quando bem apresentada, tem o poder de despertar o impulso da compra” (p. 50). Assim, Borges (2016) defende que a embalagem deve ser atrativa, para que o consumidor pegue o produto, confira seu rótulo e o adquira. Isso posto, apresentamos na sequência os procedimentos metodológicos que foram usados para o desenvolvimento desta pesquisa. E, posteriormente, apresentamos as análises de seis rótulos de embalagens de produtos para cabelos cacheados que fazem parte do corpus deste trabalho. Leitura, Maceió, n. 66, set./dez. 2020 – ISSN 2317-9945. Estudos linguísticos e literários, p. 130-145. Análise dos rótulos de embalagens de produtos capilares sob a perspectiva do leitor presumido A análise de gêneros se enriquece quando considera as práticas sociais e discursivas que envolvem o gênero a ser investigado. A fim de obter uma análise mais completa do gênero, recorremos ao contexto histórico, cultural e social que envolve os rótulos de embalagens de produtos para cabelos cacheados, analisando a pesquisa de Souza (2008), o Dossiê BrandLab: A Revolução dos Cachos e o Caderno de tendências 2019–2020 da ABIHPEC e SEBRAE). Essa análise mostrou que o gênero rótulo de embalagens de produtos capilares apresenta valorações culturais relacionadas ao tipo de cabelo considerado como padrão de beleza, e essas valorações são influenciadas pelo contexto histórico e social. Souza (2008) constatou que os rótulos apresentavam valorações culturais padronizadas em sua constituição, como “cabelo liso” e “cabelo normal”, que negavam e/ou ofuscavam, por exemplo, valorações como “cabelo crespo” ou “cabelo anormal”, inexistentes nos rótulos que analisou. Conforme a autora, as referências a cabelo não-liso tentavam “discipliná-lo”, como por exemplo, o xampu Elsève que propõe “comportar os cachos”; e concluiu que os rótulos que sugeriam que os cabelos cacheados podiam ser domados ou comportados apresentavam um discurso racista. Além desse discurso, a autora identificou também um discurso sexista, pois os rótulos se dirigiam apenas às mulheres. O Dossiê BrandLab: A Revolução dos Cachos, produzido em 2017, afirma que cresceu em 309% nos últimos dois anos o interesse por cabelos afro e “a busca por cabelos cacheados cresceu 232% no último ano, ultrapassando pela primeira vez a busca por cabelos lisos”. Esses dados apontam que no momento há uma predileção por produtos voltados para cabelos cacheados. Não é à toa que as fabricantes de cosméticos estão lotando as gôndolas dos supermercados com produtos destinados para esse tipo de cabelo. Esse dossiê indica também que “24% das mulheres de 18 a 24 anos reconhecem seu cabelo como cacheado” e que “quanto mais velha a mulher, mais difícil é declarar o cabelo como cacheado”. A partir desses dados, é possível notar que o público mais jovem é quem faz parte dessa “revolução dos cachos” e mais se interessa pelo assunto. Já o CADERNO DE TENDÊNCIAS 2019–2020 da ABIHPEC e SEBRAE apresenta um cenário diferente do que a pesquisa de Souza (2008) mostrou sobre valorações culturais referentes a cabelos e corrobora os dados desse dossiê. O caderno destaca que atualmente prevalece a preferência pelos cabelos na sua forma natural, sejam eles ondulados, cacheados, crespos ou lisos. Metodologia Esta pesquisa se classifica como qualitativa, com caráter descritivo e analítico de análise dos dados. Considerando o objetivo deste estudo de demonstrar como a figura do leitor presumido do rótulo de embalagens de produtos para cabelos cacheados/crespos é construída, montamos um corpus composto de seis rótulos de embalagens de produtos para esses tipos de cabelos, mas selecionamos apenas dois para esta análise por uma questão de espaço. Optamos por rótulos comercializados no Brasil em 2019, de duas marcas selecionadas nas redes sociais Facebook e/ou Instagram. Os rótulos de embalagens desses produtos foram coletados, e, quando possível, destacados das embalagens e, posteriormente, digitalizados ou fotografados para serem inseridos como imagens nesta pesquisa. Para realizar a análise do corpus, quando necessário, recortamos fragmentos dos rótulos para exemplificar a construção da figura do leitor nesse gênero. Optamos por identificar e segmentar o corpus referindo-nos aos rótulos de embalagens através da abreviatura RE seguida dos números 1 e 2. Para realizar este estudo partimos de alguns pressupostos bakhtinianos para o estudo do gênero, encontrados em Marxismo e Filosofia da Linguagem e Estética da Criação Verbal, enfatizando a perspectiva da formação cultural e ideológica resultante dos textos visados no pensamento bakhtiniano. Para complementar esse estudo nos apoiamos em autores representantes da abordagem sociorretórica de gêneros, como Bazerman (2006), 136 136 Leitura, Maceió, n. 66, set./dez. 2020 – ISSN 2317-9945. Estudos linguísticos e literários, p. 130-145. Miller (2009 [1984]) e Swales (1990; 2004). Buscamos explicar a construção do leitor presumido dos rótulos de embalagens de produtos para cabelos cacheados por meio de uma análise contextual, a partir de um estudo do texto voltado ao contexto histórico, cultural e social em que esse gênero é produzido. Para isso, buscamos por informações sobre o mercado de produtos capilares e as principais tendências desse mercado no Dossiê BrandLab: A Revolução dos Cachos e no Caderno de tendências 2019–2020 da ABIHPEC e SEBRAE, que se configuram como material documental coletado na internet. Para compreender o rótulo como registro histórico e traçar um breve paralelo entre os rótulos de embalagens de produtos capilares produzidos há doze anos com os produzidos atualmente, recorremos a Souza (2008). Leitura, Maceió, n. 66, set./dez. 2020 – ISSN 2317-9945. Estudos linguísticos e literários, p. 130-145. Análise dos rótulos de embalagens de produtos capilares sob a perspectiva do leitor presumido Segundo ABIHPEC e SEBRAE (2019, p. 48) cresceu a procura por produtos específicos para cabelos cacheados, crespos e afins. 137 Entendemos que isso é resultado de mudanças nos hábitos comportamentais e culturais da sociedade. No corpus analisado, no lugar da tradicional linguagem técnica dos rótulos de embalagens, prevalece o uso de uma linguagem mais informal, como é o caso da recorrência do uso da palavra você, revelando a tentativa dos produtores do texto de estabelecer uma relação de proximidade com o leitor. No RE1 o uso informal da língua já pode ser notado pela escolha do nome da linha (#todecacho). Veja-se: Figura 1: Imagem do rótulo de embalagem da Maionese Capilar da Salon Line- RE1 Figura 1: Imagem do rótulo de embalagem da Maionese Capilar da Salon Line- RE1 Figura 1: Imagem do rótulo de embalagem da Maionese Capilar da Salon Line- RE1 Fonte: Imagem reproduzida pelos autores Fonte: Imagem reproduzida pelos autores A parte central do rótulo informa que o produto é destinado para cabelos crespos e crespíssimos. As frases presentes nessa parte, como “Uma nutrição power!”, “Tchau, tchau ressecamento!” e “Nutrição de abalar!” e “Desembaraço mais que perfeito!”, também representam um uso mais informal da língua. Além disso, sugerem que o leitor, assim como o produtor do texto, é alguém que também “está de cacho”, quer nutrir seus cabelos cacheados e/ou crespos, facilitar o desembaraço deles e livrá-los do ressecamento. Da imagem acima, transcrevemos a parte introdutória (que serve para apresentar o produto) desse rótulo: MAIONESE CAPILAR # UMA NUTRIÇÃO POWER só podia ser com a nossa MAIONESE! Uma delícia de produto, mas claro, para o seu cabelo… ele vai amarrr, e você? Vai a loucuraaa! Primeiro porque o mix de óleos + proteína do ovo deixam os fios emolientes (com fácil desembaraço) e super nutridos. Sabe aquele cabelo mega ressecado? Com essa maionese isso não existe mais! Além de uma performance incrível, o cheirinho é mara e do jeito que você já ama! Afinal, ela é o terror dos cabelos ressecados… a nossa maionese é potente e turbinada! Uma máscara de balançar corações e arrasar quarteirões! Então bora?!;) No texto transcrito acima, é possível identificar que o leitor do rótulo é 138 Leitura, Maceió, n. 66, set./dez. 2020 – ISSN 2317-9945. Estudos linguísticos e literários, p. 130-145. Leitura, Maceió, n. 66, set./dez. 2020 – ISSN 2317-9945. Estudos linguísticos e literários, p. 130-145. denominado de você (duas ocorrências no texto). A opção pelo uso de pronome de tratamento mais informal sugere que o produtor do texto busca empatia, proximidade e intimidade com o leitor. O uso dos pronomes possessivos como “nossa MAIONESE” (duas ocorrências) e “seu cabelo” (uma ocorrência) também indica essa tentativa de cativar o consumidor, e, nesses casos, são usados como recursos persuasivos. Podemos observar que empresa “cede” sua voz técnica, distante, a alguém com uma voz mais leve e descontraída, para falar bem do produto em seu nome, como se estivesse batendo um papo informal com uma amiga. A utilização de perguntas retóricas (“ele vai amarrr, e você? Vai a loucuraaa!” e “Sabe aquele cabelo mega ressecado? Com essa maionese isso não existe mais!”) é também uma estratégia de interação mais direta com o leitor, na busca de empatia. Já o uso abundante de adjetivos (power, emolientes, potente, turbinada - por exemplo) e de substantivos com valor de adjetivo (por exemplo, delícia de produto) além de destacar a qualidade e revelar uma apreciação positiva do produto, sugere que o produtor do texto conhece bem o produto, já o testou e o aprovou, assim, assemelha-se a um vendedor tentando convencer um cliente de que o produto é bom, buscando despertar o desejo de um leitor que, presume-se, se preocupa com a beleza e a nutrição de seus cabelos. O convite feito no final da parte introdutória, “Então bora?!;), reforça essa ideia. Fonte: Imagem reproduzida pelos autores. Podemos observar no pedido para que o leitor curta a página da empresa nas redes sociais e poste uma foto na rede social Instagram com a hashtag #todecacho para aparecer no site www.todecacho.com.br, que o leitor visado é aquele que está conectado, tem orgulho de seus cabelos crespos ou cacheados, adora tirar fotos deles e postá-las nas redes sociais. Conforme o dossiê que subsidia essa investigação, “quanto mais velha a mulher, mais difícil é declarar o cabelo como cacheado”, assim podemos inferir que esses hábitos de exposição dos cabelos cacheados em redes sociais podem ser atribuídos principalmente a pessoas mais jovens. Há neste rótulo referência a um público-alvo específico, dentro desse grande público de consumidores de produtos para cabelos cacheados e crespos, recortamos do RE1 a imagem abaixo para analisar esse público: Figura 3: Selo de proteção aos animais. Fonte: Imagem reproduzida pelos autores. Fonte: Imagem reproduzida pelos autores. Nessa imagem está escrito: “Não testado em animais/pea.org.br”. A informação de que o produto não é testado em animais, e de que essa informação pode ser conferida no site do PEA (Projeto Esperança Animal), visa alcançar leitores que têm afinidade com a causa da proteção aos animais e que não consomem produtos de empresas de cosméticos que testam seus produtos neles. Assim, ao destacar essa informação, a empresa quer mostrar aos consumidores que também se preocupa com os animais, que os protege, por isso não realiza testes neles. Na análise da Figura 1, conseguimos compreender a perspectiva de Bakhtin (2016), a respeito do enunciado na relação com o interlocutor, considerando toda a cadeia enunciativa, de modo a ver que o enunciado precisa ser analisado a partir dessas relações, inclusive os aspectos históricos em torno dessa cadeia. O rótulo visto foi produzido considerando historicamente a quebra de um discurso em que os produtos dessa natureza eram marcados pelo uso de termos preconceituosos, como pontuamos na análise, e que agora assumem um posicionamento de respeito e proximidade quanto ao interlocutor/consumidor, voltando, dessa maneira, para a perspectiva do leitor presumido apresentada nesta pesquisa. Antes de prosseguirmos com as análises, é importante fazermos a mesma ressalva feita por Alves Filho e Soares de Sá (2015) de que apenas pressupomos o leitor/público-alvo, isto é, apenas criamos a expectativa de leitores, já que os leitores reais, os que de fato terão acesso à mensagem, “podem ultrapassar a fronteira daquilo que foi inicialmente presumido” (p. 645). MAIONESE CAPILAR O prolongamento de consoantes e vogais (amarrr e loucuraaa), por exemplo, comprova o uso de recursos linguísticos inovadores nos rótulos de embalagens, que também servem para avaliar o produto e ao mesmo tempo buscar a empatia do leitor (a linguagem metonímica e com traços de personificação presente no texto do rótulo tenta atraí-lo. Em “seu cabelo… ele vai amarrr”, sabemos que o cabelo não sente amor por nada, mas seu dono sim. Dessa forma, na verdade quem vai amar o produto é o leitor que usá-lo). Notamos também o uso informal da língua na frase “o cheirinho é mara e do jeito que você já ama”. A utilização recorrente da palavra “ama” e a abreviação da palavra maravilhoso (mara), bastante usada, por exemplo, por blogueiras e, principalmente por jovens, sugerem que o público leitor desse rótulo é jovem, conectado à internet e às redes sociais. O trecho que aparece abaixo da parte introdutória também pressupõe um leitor mais jovem. Veja-se: Leitura, Maceió, n. 66, set./dez. 2020 – ISSN 2317-9945. Estudos linguísticos e literários, p. 130-145. 13 Figura 2: Reprodução de um trecho da parte posterior do rótulo. Curta! /todecacho /todecachobrasil @todecacho @todecacho Poste uma foto no Instagram com a hashtag #todecacho e apareça no nosso site: www.todecacho.com.br Figura 2: Reprodução de um trecho da parte posterior do rótulo. /todecachobrasil 139 Leitura, Maceió, n. 66, set./dez. 2020 – ISSN 2317-9945. Estudos linguísticos e literários, p. 130-145. Leitura, Maceió, n. 66, set./dez. 2020 – ISSN 2317-9945. Estudos linguísticos e literários, p. 130-145. Fonte: Imagem reproduzida pelos autores. No RE2, o registro informal de uso da língua também é notado: 140 140 Leitura, Maceió, n. 66, set./dez. 2020 – ISSN 2317-9945. Estudos linguísticos e literários, p. 130-145. Figura 4: Imagem da frente e do verso do Rótulo de embalagem do Shampoo Hidratante “Meu cacho minha vida” da Lola Cosmetics – RE2. Fonte: Imagem fotografada pelos autores. Fonte: Imagem fotografada pelos autores. No texto que consta na parte frontal do rótulo do produto “Meu cacho minha vida” há a seguinte apresentação do produto: #VocêPediuaLolaAtendeu #CriseTáTendo Leitura, Maceió, n. 66, set./dez. 2020 – ISSN 2317-9945. Estudos linguísticos e literários, p. 130-145. #VocêPediuaLolaAtendeu As palavras sublinhadas nele revelam que as leitoras visadas são tratadas pela produtora do texto como fãs da marca Lola (Loletes Curlies significa Loletes encaracoladas) e que o xampu apresentado no rótulo será o amante (o lover) dessas fãs da Lola. Assim como o RE1, o rótulo desse xampu também destaca que a empresa não faz teste em animais. A expressão em inglês Cruelty free, que no rótulo do xampu aparece embaixo do desenho de um ratinho, significa “livre de crueldade” e se aplica a produtos que não foram testados em animais. Destaca-se também, embaixo do desenho de uma ovelhinha, que o produto é go vegan, o que significa que o cosmético produzido não possui ingredientes de origem animal e nem foi testado em animais. Dessa forma, o texto visa alcançar um público feminino que, assim como a empresa, adota o veganismo como filosofia de vida, isto é, que não usa nem explora animais em suas atividades. No final do rótulo, há ainda o destaque para a informação de que o xampu é “LIVRE DE: Glúten. Sulfato. Corantes. Sintéticos. Ftalatos. OGMs. Silicone. Sal. Parabenos. Óleo Mineral. Parafina. Derivados de animais.” Essas informações buscam atingir também as leitoras que são adeptas das técnicas No Poo e Low Poo (Técnica de lavagem do cabelo sem uso, ou com uso bastante reduzido, de xampu e/ou produtos que tenham sulfatos e parabenos). Os rótulos de embalagens de produtos para cabelos cacheados analisados se dirigem a um público em sua maioria feminino, buscando, através de uma linguagem única e intimista, uma proximidade com esse público. Observamos que a empresa “empresta” sua voz a uma pessoa que se comunica em tom de conversa informal, descontraída e animada com o leitor. Portanto, ao menos nesses rótulos, notamos que o leitor presumido faz parte de um público feminino jovem, que sabe o que quer e o que não quer encontrar em produtos para cabelos cacheados e que é tratado como uma amiga particular, com quem a empresa fala pessoalmente, num clima de intimidade. Podemos aqui marcar Bakhtin (2006), na visão de Pereira e Rodrigues (2014), de que a ideologia refrange os discursos. Os valores sociais são sustentados pela ideologia. Podemos ver claramente uma ideologia marcada nos discursos presentes nos rótulos analisados, como já sublinhamos. Além disso, conseguimos observar a defesa bakhtiniana a respeito da cadeia que deve ser considerada em torno dos discursos. #VocêPediuaLolaAtendeu Miga, chega de chororô! Tá tendo crise sim, mas tá tendo aquela velha zuêira também porque ninguém é de ferro, né? E como somos empáticas, empoderadas and sacadoras dos paranauês de crise, fizemos um produto bem bapho para você ostentar neste momento difícil dificílimo. #CriseTáTendo #EmpatiaTambém #LolaEmpoderadaNaCrise #LolaEmpoderadaNaCrise Nesse trecho, determina-se claramente o grupo social para o qual o texto é construído: a interpelação carinhosa revelada pela palavra miga mostra que o público visado é o feminino. Os termos negritados também revelam um tratamento informal com a leitora (uso do pronome você, de variedades linguísticas como “tá” e as gírias), a tentativa da produtora do texto (que também assume uma voz feminina, como revela a expressão “somos empáticas”) de estabelecer uma intimidade, proximidade e preocupação com a leitora no momento de crise (qualificado como “momento difícil dificílimo”) vivido pelo país no período em que o produto foi lançado, agosto de 2016. Desse modo, a empresa tenta mostrar-se preocupada com a leitora que nesse momento de crise clamava por produtos com preços mais acessíveis (como mostra o trecho #VocêPediuaLolaAtendeu). Da parte de trás da embalagem, analisemos os seguintes trechos e recorte de elementos imagéticos: O que é: Shampoo Hidratante de uso diário para nossas Loletes Curlies. O óleo de patauá e os extratos vegetais presentes na fórmula promovem suavidade e maciez aos cabelos, aumentando a sua umidade natural e elasticidade. 141 141 Leitura, Maceió, n. 66, set./dez. 2020 – ISSN 2317-9945. Estudos linguísticos e literários, p. 130-145. O que faz: Não importa quantas vezes você lava seu cabelo, mas o fato é que o shampoo é o primeiro passo para manter seus cachos saudáveis, limpos e vibrantes. Nosso Shampoo Hidratante Meu Cacho Minha Vida é suave e emoliente. Fica a dica: Shampoos tradicionais tiram a umidade natural dos fios, por isso o Shampoo Meu Cacho Minha Vida é o mais adequado para cabelos crespos e cacheados devido aos seus ingredientes suaves e fórmula não agressiva. Ele será o lover de nossas Loletes Curlies. Figura 5: Trecho da parte posterior inferior do rótulo. Fonte: Imagem reproduzida pelos autores. Figura 5: Trecho da parte posterior inferior do rótulo. Figura 5: Trecho da parte posterior inferior do rótulo. Fonte: Imagem reproduzida pelos autores. Fonte: Imagem reproduzida pelos autores. Nesse trecho, o uso da linguagem informal é menos marcante, mas está presente. Leitura, Maceió, n. 66, set./dez. 2020 – ISSN 2317-9945. Estudos linguísticos e literários, p. 130-145. #VocêPediuaLolaAtendeu Em outras palavras, precisamos entender que esses discursos são dirigidos para um interlocutor, que é marcado historicamente. Como Alves 142 142 Filho e Soares de Sá (2015, p. 642) delineiam, os gêneros refletem a sociedade de uma época. Sendo assim, podemos compreender quais características construíram determinado grupo social a partir da análise dos gêneros que refletem aquele período. Os autores marcam que essa visão nos leva à compreensão de que “os gêneros do discurso suscitam em seus usuários aquilo que se pode chamar de expectativa de entendimento”, o que Bakhtin denominou de “auditório social” e que aqui estamos marcando como “leitor presumido”, conforme o delineamento teórico ora suscitado. Esse leitor presumido é identificado pelas marcas que pontuamos a partir da análise dos dois exemplares do corpus apresentado nesta pesquisa. Conforme ABIHPEC e SEBRAE (2019), o mercado está investindo alto em produtos para cabelos cacheados, pois esse é um dos tipos de cabelos naturais que as mulheres estão assumindo e desejam produtos específicos para eles. Constatamos que em nenhum dos rótulos do nosso corpus aparece termos como “cachos disciplinados/comportados”, encontrados por Souza (2008). Isso demonstra que não há, nos rótulos de embalagens produzidos no contexto atual, espaço para um discurso racista que elege como padrão de beleza apenas o cabelo liso, como o identificado por Souza (2008). Esse é um indício de que as transformações sociais se refletem nos gêneros, e estes acompanham o dinamismo social. Considerações finais A análise empreendida revelou que o leitor presumido dos rótulos de embalagens de produtos destinados para cabelos cacheados é composto principalmente por mulheres jovens, conscientes do que querem e do que não querem encontrar nesses produtos, e que estão conectadas às redes sociais. Inseridos na esfera de circulação social da produção e do consumo, os rótulos de embalagens de produtos capilares são produções que fazem parte do cotidiano das pessoas, de suas práticas sociais. Para atingir seu objetivo de informar e promover os produtos fabricados os produtores dos textos dos rótulos de embalagens, precisam conhecer bem o público-alvo a que seus produtos se destinam. Esperamos que esta pesquisa contribua para suprir a carência de uma investigação mais acurada sobre a forma/função do gênero rótulo de embalagens de produtos capilares. Reconhecemos, porém, que este estudo apresenta limitações, seja pela restrição da amostra, seja pelo enfoque maior nos seus elementos verbais. Visto que se limitou à análise de apenas um tipo de público dos rótulos de embalagens de produtos capilares, os quais, como sabemos, têm público bem diverso, sugerimos que novas pesquisas em torno desse mesmo objeto de estudo incluam, por exemplo, a análise comparativa de diferentes públicos-alvo dos rótulos. Referências 143 143 Leitura, Maceió, n. 66, set./dez. 2020 – ISSN 2317-9945. Estudos linguísticos e literários, p. 130-145. dez. 2019. ALVES FILHO, F; SOARES DE SÁ, S. O leitor presumido nos anúncios de carros veiculados em jornais impressos de Teresina (PI) de 1950 a 2002. Forum linguistic, Florianópolis, v. 12, n.2, p.637- 652, abr./jun, 2015. ANVISA. Agência Nacional de Vigilância Sanitária. Resolução da diretoria colegiada - RDC Nº 259, de 20 de setembro de 2002. Disponível em: http://portal.anvisa.gov.br/documents/33880/2568070/RDC_259_2002.pdf/e40c2ecb- 6be6-4a3d-83ad-f3cf7c332ae2. Acesso em: 24 mai. 2020. ANVISA. Agência Nacional de Vigilância Sanitária. Resolução da diretoria colegiada - RDC Nº 07, de 10 de fevereiro de 2015. Dispõe sobre os requisitos técnicos para a regularização de produtos de higiene pessoal, cosméticos e perfumes. Disponível em: http://portal.anvisa.gov.br/documents/10181/2867685/RDC_07_2015_.pdf/. Acesso em: 29 abr. 2020. BAKHTIN, M. Estética da Criação Verbal. Tradução Paulo Bezerra. São Paulo: Martins Fontes, 2003 [1979]. BAKHTIN, M. Marxismo e filosofia da linguagem. 14. ed. Tradução Paulo Bezerra. São Paulo: Hucitec, 2006 [1929-1930]. BAKHTIN, M. Os gêneros do discurso. 1. ed. Organização, tradução, posfácio e notas de Paulo Bezerra; notas da edição russa de Serguei Botcharov. São Paulo: Editora 34, 2016. BAKHTIN, M./VOLOSHINOV, V. N. Discurso na vida e discurso na arte (sobre poética sociológica)”. (tradução para o português feita por Carlos Alberto Faraco e Cristovão Tezza, para uso didático, tomou como base a tradução inglesa de I. R. Titunik (“Discourse in life and discourse in art – concerning sociological poe-tics”), publicada em V. N. Voloshinov, Freudism, New York. Academic Press, 1976 [1926]. BAZERMAN, C. Gêneros, Agência e Escrita. Judith C. Hoffnagel; Ângela P. Dionísio (orgs). São Paulo: Cortez, 2006. BIASI-RODRIGUES, B. A diversidade de Gêneros textuais no ensino: um novo modismo? In: PERSPECTIVA, Florianópolis, v.20, n.01, p.49-64, jan./jun, 2002. BORGES, R. B. de O. A tradução de rótulos virtuais no e-commerce: um olhar sobre cosméticos infantis. 2016. 147 f. Dissertação (Mestrado em Estudos da Tradução). Departamento de Línguas Estrangeiras e Tradução, Universidade de Brasília, Brasília, 2016. BRASIL. Lei nº. 6.360, 23 de setembro de 1976. Disponível em http://www.planalto.gov.br/ccivil_03/LEIS/L6360.htm#art5. Acesso em: 09 jun. 2020. KOCH, I. G. V. O texto e a construção dos sentidos. 10. ed., 4. reimp. São Paulo: Contexto, 2016. MARCUSCHI, L. A. Produção textual, análise de gêneros e compreensão. São Paulo: Parábola Editorial, 2008. 144 Leitura, Maceió, n. 66, set./dez. 2020 – ISSN 2317-9945. Estudos linguísticos e literários, p. 130-145. 144 MILLER, C. R. Gênero como ação social. Leitura, Maceió, n. 66, set./dez. 2020 – ISSN 2317-9945. Estudos linguísticos e literários, p. 130-145. Referências In: Estudos sobre: gênero textual, agência e tecnologia. Tradução de Judith ChamblissHoffnagel et al. Recife: Ed. Universitária da UFPE. p. 21-44, 2009 [1984]. PEREIRA, Rodrigo Acosta; RODRIGUES Rosângela Hammes. O conceito de valoração nos estudos do círculo de Bakhtin: a inter-relação entre ideologia e linguagem. Linguagem em (Dis)curso – LemD, Tubarão, SC, v. 14, n. 1, p. 177-194, jan./abr. 2014. SOARES DE SÁ, S. As alterações ocorridas nos anúncios de carros em jornais impressos de Teresina (1950 a 2002). Dissertação (Mestrado em Letras). Universidade Federal do Piauí, Teresina, 2013. SOUZA, C. A. Lima de. Alguns rótulos de produtos de higiene pessoal: Gênero Discursivo e(em) Estudos da Cultura. 2008. 163 f. Dissertação (Mestrado em Letras) – Programa de Mestrado em Letras, Universidade Federal de São João del-Rei, São João del- Rei, 2008. SWALES, J. M. Genre Analysis: English in Academic and Research Settings. Cambridge (UK); New York: Cambridge University Press, 1990. SWALES, J. M. Research genres: explorations and applications. New York: Cambridge University Press, 2004. THINK WITH GOOGLE. Dossiê BrandLab: A Revolução dos Cachos. Disponível em: https://www.thinkwithgoogle.com/intl/pt-br/advertising- channels/v%C3%ADdeo/revolucao-dos-cachos/Acesso em: 09 jun. 2020. 145 145 Leitura, Maceió, n. 66, set./dez. 2020 – ISSN 2317-9945. Estudos linguísticos e literários, p. 130-145. Leitura, Maceió, n. 66, set./dez. 2020 – ISSN 2317-9945. Estudos linguísticos e literários, p. 130-145.
https://openalex.org/W4293347595	https://zenodo.org/records/7026543/files/SpatHum2022_paper_14.pdf	English	null	A G eospatial and C artographic A nalysis of the Galician B lack G enre: the M ap as a T ool for a S ociology of S pace and for L iterary E ducation	Zenodo (CERN European Organization for Nuclear Research)	2,022	cc-by	618	A Geospatial and Cartographic Analysis of the Galician Black Genre: the Map as a Tool for a Sociology of Space and for Literary Education If space is, in general, a key pillar of narratology, in the case of crime novels the relevance and load of meaning of space is particularly relevant. In the noir genre, spaces often convey symbolic and sociological values, are intimately linked to the narrative plot and constitute more than a mere spatial framework. Often the crime takes the form of a message, and the investigator presents himself as a hermeneutist who reads the signs inscribed in space. This spatial relevance is also revealed in the fact that there are perceptible geographical marks in the functioning of the genre, as can be exemplified by the emergence of the Nordic crime novel or by the role played by the big city, as well as by the frequent presence of symbolic spaces pregnant with a semiotic charge such as islands. Starting from the hypothesis that the noir genre constitutes a privileged vehicle for the expression of geospatial tensions, we intend to analyze, using geographic information systems (GIS), the functioning of space in the Galician noir novel, as well as in audiovisual fiction. Significantly, in recent years the black genre has emerged strongly in the Spanish state precisely in peripheral spaces with a culture and language of its own in which identity tensions between social imaginaries are enormously enhanced. Thus, the trilogy of Baztán by Dolores Redondo and the cycle of La ciudad Blanca (The White City) by Eva G. Sáenz de Urturi have achieved enormous diffusion, to which transmediality has also contributed through film adaptations. It is no coincidence that in both series the murders take on a ritual value that entails a journey through emblematic spaces and traditions of Basque and Navarrese culture. The resolution of crimes involves delving into cultural elements, spaces and meanings and establishes a code that remains opaque for those who do not know the geographical and cultural keys. Beyond the purpose of disseminating and preserving cultural traditions and values, both series point to the centrality of space and the social imaginary in the genre. In fact, it could be said that the rise of the noir genre in recent years has a direct and not accidental connection with the spatial turn that characterizes the postmodern chronotope. p The emergence of the Galician noir novel is late. Crime en Compostela (1984) by Carlos Reigosa is considered the work that marks the beginning of the genre in Galicia. A Geospatial and Cartographic Analysis of the Galician Black Genre: the Map as a Tool for a Sociology of Space and for Literary Education But in recent years we can speak of a real hatching. And while in the Galician literary tradition the primacy corresponded to the rural area, the noir genre has privileged the urban environment, becoming a true producer of urban literary geographies. Faced with the inaugural presence of Santiago de Compostela, Vigo has emerged as the city in which black fiction is set in a preferential way in Galicia. Faced with the impossibility of carrying out a complete emptying of all the geographical references contained in the Galician noir fictions, we will choose to capture on maps the priority spaces of setting (geolocation of the crime, investigation spaces...) that allow us to have a cartographic tool for the interpretation of social and territorial meanings. By developing these maps we hope to illustrate the potential of literary cartography to generate useful tools for the analysis and understanding of the ideological, identity, and sociological mechanisms present in the conformation of literary spaces, as well as to explore its potential for literary dissemination and education.
https://openalex.org/W4392102792	https://www.actbio-vsuet.ru/jour/article/download/5041/4553	Russian	null	ПЕРСПЕКТИВЫ АКУСТИЧЕСКИХ СЕНСОРНЫХ СИСТЕМ ДЛЯ ОПРЕДЕЛЕНИЯ АНТИБАКТЕРИАЛЬНЫХ ПРЕПАРАТОВ	Deleted Journal	2,023	cc-by	1,151	О.И. Гулий1, Б.Д. Зайцев2, О.А. Караваева1, А.В. Мартыненко3, И.А. Бородина2 О.И. Гулий , Б.Д. Зайцев , О.А. Караваева , А.В. Мартыненко , И.А. Бородина Институт биохимии и физиологии растений и микроорганизмов, ФИЦ «Саратовский научный центр РАН» (ИБФРМ РАН Саратов, 410049, Россия биохимии и физиологии растений и микроорганизмов, ФИЦ «Саратовский научный центр РАН» (ИБФРМ Р Саратов, 410049, Россия Институт радиотехники и электроники им. В.А. Котельникова РАН, Саратовский филиал, Саратов, 410019, Россия Саратовский национальный исследовательский государственный университет имени Н.Г. Чернышевского, Саратов, 410012, Россия Широкая распространенность инфекционных заболеваний обусловливает активное применение антимикробных лекарственных средств, среди которых наиболее значимыми являются антибактериальные препараты. Применение антибиотиков стало настоящей революцией в лечении инфекционных заболеваний, но в дальнейшем это привело не только к появлению антибактериальных препаратов и продуктов их деградации в объектах окружающей среды, но и к развитию антибиотикорезистентности бактерий (способность бактерий адаптироваться к воздействию антимикробных препаратов). Появление антибиотиков в окружающей среде (особенно в водной среде) стимулирует развитие новых методов контроля антибактериальных препаратов в продуктах питания, сточных водах фармацевтических предприятий и других объектах. Для определения антибиотиков применяют микробиологические, спектрофотометрические, флуориметрические, хемилюминесцентные, различные варианты хроматографических методов, в том числе высокоэффективную жидкостную хроматографию и хромато-масс-спектрометрию, инверсионную вольтамперометрию, электроаналитическое определение с модифицированными электродами, а также биосенсорные методы. Биосенсорные методы анализа активно развиваются в последнее время и являются неотъемлемой частью экологического мониторинга окружающей среды. Одним из перспективных направлений сенсорных методов определения антибиотиков являются акустические сенсорные системы. Акустические датчики, будучи очень чувствительными, имеют преимущество перед датчиками других типов, поскольку они могут обнаруживать изменения как механических параметров (плотность, упругость, вязкость), так и электрических свойств (диэлектрическая проницаемость, проводимость) среды, в отличие от других микросенсорных устройств, которые следят за изменением только одного физического параметра. Акустические датчики измеряют изменения в характеристиках акустических волн (таких как скорость, затухание, частота) при их распространении в пьезоэлектрическом кристалле, контактирующем с исследуемой средой. Акустические биосенсоры используют пьезоактивные акустические волны для создания аналитического сигнала. Принцип действия электроакустических методов анализа основан на регистрации биоспецифических реакций в жидкой суспензии, контактирующей с поверхностью пьезоэлектрического звукопровода, по которому распространяется пьезоактивная акустическая волна. В последнее время для создания акустических биосенсоров широко используются пьезоэлектрические резонаторы или линии задержки с распространяющейся поверхностной или пластинчатой акустической волной. Такие сенсоры чувствительны к изменению механических или электрических свойств биологического объекта, контактирующего с поверхностью звукопровода. Акустические биосенсоры чаще всего выполнены на основе таких пьезоэлектрических кристаллов, как кварц, ниобат лития, или танталат лития, поскольку они характеризуются высокой химической устойчивостью. Акустические волны, возбуждаемые в пьезоэлектрической среде, позволяют создать целое семейство датчиков, характеризующихся высокой чувствительностью, быстротой проведения анализа, дешевизной, и небольшими размерами [1]. Актуальная биотехнология №1, 2023 УДК 579.6 Актуальная биотехнология №1, 2023 УДК 579.6 Актуальная биотехнология №1, 2023 УДК 579.6 Актуальная биотехнология https://doi.org/10.20914/2304-4691-2023-1-8-9 Актуальная биотехнология №1, 2023 Актуальная биотехнология №1, 2023 Актуальная биотехнология На основе резонатора с поперечным электрическим полем разработан компактный акустический анализатор для экспресс-анализа антибиотиков на примере хлорамфеникола в водном растворе. Анализирующая часть устройства включает цифровой генератор сигнала и управляющий микроконтроллер, который соединяется с персональным компьютером. В качестве сенсорного элемента использовали бактериальные клетки, чувствительные к определяемому антибиотику. Установлена корреляция экспериментальных данных, полученных с помощью акустического датчика с результатами, полученными с помощью световой фазово-контрастной микроскопии и стандартного микробиологического анализа. Представленный способ демонстрирует стабильность, чувствительность и повторяемость результатов [5]. Биологические тест-системы на основе пьезоэлектрического резонатора с поперечным электрическим полем с использованием бактерий в качестве сенсорного элемента являются весьма перспективными для экспресс-анализа антибиотиков и позволяют определять низкие концентрации антибиотика непосредственно в жидкости без необходимости иммобилизации компонентов анализа. Дополнительным преимуществом предлагаемых датчиков является возможность их многократного использования, из-за чего отсутствует необходимость в подборе условий для утилизации тест-систем после проведения анализа. Дальнейшая стандартизация и автоматизация акустических сенсорных систем позволит расширить круг их применения и использования в микробиологии, биотехнологии, ветеринарии, медицине, защите окружающей среды. Исследование выполнено за счет гранта Российского научного фонда № 22-29-00587. Исследование выполнено за счет гранта Российского научного фонда № 22-29-00587. 3. Guliy О.I., Zaitsev B.D., Semyonov A.P., Alsowaidi A.К.М., Teplykh A.A., Karavaeva О.А., Borodina I.A. Microbial acoustic sensor test-system based on a piezoelectric resonator with a lateral electric field for kanamycin detection in liquid. Ultrasonics 2022, 120, 106651. https://doi.org/10.1016/j.ultras.2021.106651. , p g j 4. Гулий О.И., Зайцев Б.Д., Шихабудинов А.М., Бородина И.А., Ларионова О.С., Жничкова Е.Г. Определение чувствительности микробных клеток к полимиксину методом электроакустического анализа. Антибиотики и химиотерапия 2017, 62 (3–4), 3–9. doi 10.24411/0235–2990–2017–00031. О.И. Гулий1, Б.Д. Зайцев2, О.А. Караваева1, А.В. Мартыненко3, И.А. Бородина2 До недавнего времени акустические датчики не применялись для определения антибиотиков и первое упоминание о возможности их применения для оценки воздействия антибактериальных препаратов на бактерии приведено в работе [2]. Перспективность применения датчика на основе резонатора с поперечным электрическим полем с помощью микробных клеток для определения антибиотиков непосредственно в жидкости показана на примере канамицина [3] и полимиксина [4]. 8 _ 2. Gruhl, F.J.; Länge, K. Surface acoustic wave (SAW) biosensor for rapid and label-free detection of penicillin G in milk. Food Anal. Methods 2014, 7, 430–437. doi: 10.1007/s12161–013–9642–4. ( ) 5. Zaitsev B., Borodina I., Alsowaidi A., Karavaeva O., Teplykh A., Guliy O. Microbial Acoustical Analyzer for Antibiotic Indication. Sensors 2022, 22, 2937. https://doi.org/10.3390/s22082937. 1. Gözde Durmu¸sa, N.; Linb, R.L.; Kozbergc, M.; Dermicid, D.; Khademhosseinie, A.; Demirci, U. Acoustic-based biosensors. In Encyclopedia of Microfluidics and Nanofluidics; Springer Science + Business Media: New York, NY, USA, 2014. doi 10.1007/978– 3–642–27758–0 10–2. Литература 1. Gözde Durmu¸sa, N.; Linb, R.L.; Kozbergc, M.; Dermicid, D.; Khademhosseinie, A.; Demirci, U. Acoustic-based biosensors. In Encyclopedia of Microfluidics and Nanofluidics; Springer Science + Business Media: New York, NY, USA, 2014. doi 10.1007/978– 3–642–27758–0 10–2. 1. Gözde Durmu¸sa, N.; Linb, R.L.; Kozbergc, M.; Dermicid, D.; Khademhosseinie, A.; Demirci, U. Acoustic-based biosensors. In Encyclopedia of Microfluidics and Nanofluidics; Springer Science + Business Media: New York, NY, USA, 2014. doi 10.1007/978– 3–642–27758–0 10–2. 1. Gözde Durmu¸sa, N.; Linb, R.L.; Kozbergc, M.; Dermicid, D.; Khademhosseinie, A.; Demirci, U. Acoustic-based biosensors. In Encyclopedia of Microfluidics and Nanofluidics; Springer Science + Business Media: New York, NY, USA, 2014. doi 10.1007/978– 3–642–27758–0 10–2. _ 2. Gruhl, F.J.; Länge, K. Surface acoustic wave (SAW) biosensor for rapid and label-free detection of penicillin G in milk. Food Anal. Methods 2014, 7, 430–437. doi: 10.1007/s12161–013–9642–4. _ 2. Gruhl, F.J.; Länge, K. Surface acoustic wave (SAW) biosensor for rapid and label-free detection of penicillin G in milk. Food Anal. Methods 2014, 7, 430–437. doi: 10.1007/s12161–013–9642–4. 3. Guliy О.I., Zaitsev B.D., Semyonov A.P., Alsowaidi A.К.М., Teplykh A.A., Karavaeva О.А., Borodina I.A. Microbial acoustic sensor test-system based on a piezoelectric resonator with a lateral electric field for kanamycin detection in liquid. Ultrasonics 2022, 120, 106651. https://doi.org/10.1016/j.ultras.2021.106651. 3. Guliy О.I., Zaitsev B.D., Semyonov A.P., Alsowaidi A.К.М., Teplykh A.A., Karavaeva О.А., Borodina I.A. Microbial acoustic sensor test-system based on a piezoelectric resonator with a lateral electric field for kanamycin detection in liquid. Ultrasonics 2022, 120, 106651. https://doi.org/10.1016/j.ultras.2021.106651. p g j 4. Гулий О.И., Зайцев Б.Д., Шихабудинов А.М., Бородина И.А., Ларионова О.С., Жничкова Е.Г. Определение чувствительности микробных клеток к полимиксину методом электроакустического анализа. Антибиотики и химиотерапия 2017, 62 (3–4), 3–9. doi 10.24411/0235–2990–2017–00031. 5. Zaitsev B., Borodina I., Alsowaidi A., Karavaeva O., Teplykh A., Guliy O. Microbial Acoustical Analyzer for Antibiotic Indication. Sensors 2022, 22, 2937. https://doi.org/10.3390/s22082937. post@actbio-vsuet.ru 9
https://openalex.org/W2059762258	https://europepmc.org/articles/pmc3443207?pdf=render	English	null	The Pace of Cultural Evolution	PloS one	2,012	cc-by	7,579	Introduction can be transmitted from parents to offspring, it is also transmitted obliquely, between non-parents from a previous generation, and horizontally, between contemporaries. This transmission mode gives cultural evolution the potential to spread rapidly in a population, much like an epidemic disease [3,5,7,15,17]. Humans dominate the earth’s ecosystems [1]. Today, our uncommonly large range encompasses most of the world’s terrestrial habitats, and human populations thrive in environments as diverse as the Amazonian jungle and the Arctic desert. This adaptive radiation has been explained by our capacity to socially learn information (culture) [2–4]. Culture is an inheritance system that parallels and interacts with the genetic system [5–8]. Cultural variation and innovations accumulate in a population throughout time, allowing for complex cultural adaptations to evolve [9–13]. Because it is assumed that cultural evolution occurs faster than biological evolution on average, humans can adapt to new ecosystems more rapidly than other animals [4]. Yet, the evidence for the hypothesis that cultural evolution is faster than biological evolution is anecdotal [3,14] and there are no systematic comparisons of cultural and biological rates of change. Moreover, we do not know how much faster, if at all, culture can change compared to biological phenotypes. However, it is not entirely obvious that cultural evolution is faster than biological evolution. On the one hand, the archaeo- logical record is full of instances where traditions have remained remarkably stable over hundreds of years. Microlithic tools, for example, appeared in Northern Asia around 17–18,000 Before Present (BP), and remained part of the hunter-gatherers toolkit until after 14,000 BP [18]. In addition, the Japanese sword, which is a much more complex technology, has been fabricated following essentially the same steps for nearly 700 years [19,20]. On the other hand, biologists regularly observe evolutionary change over much smaller time scales. Darwin’s Finches, a group of bird species inhabiting the Galapagos Islands, undergo morphological change on a yearly scale in what has become a textbook, classic example of biological evolution [21]. These examples indicate that the distributions of biological and cultural rates of change are, at the very least, overlapping ones. Culture might be less constrained than biology and have the potential to change instantaneously. However, much of what we know from anthropological and psychological research tells us that culture will rarely change instantly. Abstract Today, humans inhabit most of the world’s terrestrial habitats. This observation has been explained by the fact that we possess a secondary inheritance mechanism, culture, in addition to a genetic system. Because it is assumed that cultural evolution occurs faster than biological evolution, humans can adapt to new ecosystems more rapidly than other animals. This assumption, however, has never been tested empirically. Here, I compare rates of change in human technologies to rates of change in animal morphologies. I find that rates of cultural evolution are inversely correlated with the time interval over which they are measured, which is similar to what is known for biological rates. This correlation explains why the pace of cultural evolution appears faster when measured over recent time periods, where time intervals are often shorter. Controlling for the correlation between rates and time intervals, I show that (1) cultural evolution is faster than biological evolution; (2) this effect holds true even when the generation time of species is controlled for; and (3) culture allows us to evolve over short time scales, which are normally accessible only to short-lived species, while at the same time allowing for us to enjoy the benefits of having a long life history. Citation: Perreault C (2012) The Pace of Cultural Evolution. PLoS ONE 7(9): e45150. doi:10.1371/journal.pone.0045150 Received March 29, 2012; Accepted August 17, 2012; Published September 14, 2012 rles Perreault. 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. opyright: 2012 Charles Perreault. This is an open-access article distributed under the terms of the Creative Commons Attribution restricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The work was supported by a Social Sciences and Humanities Research Council of Canada Doctoral Fellowship [752-2006-2301] http://www.sshrc-crsh. gc.ca/home-accueil-eng.aspx. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The author has declared that no competing interests exist. * E-mail: cperreault@santafe.edu Charles Perreault* Charles Perreault* Santa Fe Institute, Santa Fe, New Mexico, United States of America September 2012 \| Volume 7 \| Issue 9 \| e45150 PLOS ONE \| www.plosone.org Results A linear mixed model shows that cultural rates are also inversely correlated with the time interval over which they are measured (Fig. 2, Table 2). As we study older periods of our history, taphonomic processes cause the measured time intervals to increase [46]. Cultural rates are therefore inversely correlated with their absolute age (Fig. 3, Table 3). This correlation could potentially account for one of the most salient features of the archaeological record: the pace of change of human material culture appears faster in more recent periods than it does for older ones. Biological rates are clustered by studies, taxonomic units, subpopulations (if relevant), and traits. Cultural rates are clustered by studies, technologies, and traits. A single variable, named study- series, was created and assigned the same unique ID number for the rates belonging to the same study/taxonomic unit/population/ trait series or the same study/technology/trait series. To test for the interaction between the type of rates (biological or cultural) or rate and time intervals (or rate ages), a linear mixed model of interaction was used. Time interval was assigned as a fixed effect and study-series as a random effect using an unstructured covariance matrix. The linear mixed model controls for the fact that rates are unevenly distributed among the different studies. The difference between cultural and biological rates remains significant even when the effect of the measurement time intervals is controlled (Table 2). We can use the linear mixed model that describes each type of rate to compare them on the same temporal scale. The models converge at the time scale of approximately 1 month (i.e., Dt = (1.2661027)/106 years), at which point the characteristic rate of change of both biological and cultural change is approximately 76,104 d. This corresponds to a ratio between the initial and final value of the attribute undergoing evolution (x2/x1) of 1.01. In other words, at that monthly time scale, both biological and cultural traits will show, on average, a change in their metric attributes of approximately 1%. At the scale of a one-year interval, animal morphologies change at a typical rate of 14,707 d (x2/x1 = 1.014), compared to 21,989 d (x2/ x1 = 1.022) for archaeological technologies. Cultural change is already recognizably faster at this time scale. Materials and Methods g g g However, a comparison of rates, as presented in Figure 1, is complicated by the fact that rates of biological evolution are inversely correlated with the time interval over which change is measured [2,8,28,38–41]. Evolution appears to operate faster when observing short intervals of time compared to long intervals of time. Two main reasons for this dependency have been suggested [28,38–40,42–45]. First, as the time interval of observation increases, it becomes more likely that the net rate observed is in fact averaged over several disparate rates and evolutionary reversals. This effect is in part driven by the fact that the taxonomic level of the data is likely to increase with the observation time interval. This is true for both biological and cultural evolution. The level of taxonomic units impacts rates of evolution because the evolutionary change that occurs below a taxonomic level (i.e. below the family level) is ignored. Second, rates have the time interval (Dt) in the denominator and are therefore proportional to 1/Dt. However, because of functional constraints and the effect of stabilizing selection, morphologies rarely change in proportion to Dt because they eventually reach evolutionary stasis. In contrast, Dt is unbounded and free to vary. Regardless of the mechanisms that drive it, it is necessary to control for the inverse correlation with Dt to compare groups of rates. This is accomplished by plotting the rates on an ln-rate versus ln-time interval graph, and using the linear model that best describes the correlation to compare the groups of rates on the same temporal scale [44]. Biological rates were obtained from previously analyzed data sets [28,29]. Biological rates are calculated from observations made at various taxonomic levels, ranging from subpopulations of the same species to genus and family. Cultural rates were compiled from the archaeological literature and are described in Table 1. Cultural rates are based on observations made at the level of taxonomic unit that archaeologists often refer to, albeit haphaz- ardly [30] as types, classes, and style; that is, artifacts that share a combination of attributes and that have distinct spatial and temporal distribution. Only rates corresponding to a change in one-dimensional metric attributes were collected. Biological rates observed in a laboratory or human-perturbed settings, as well as those measuring divergence between contemporary sister popula- tions, were excluded. Introduction Deviation from a group’s social norms can be costly, and can result in punishment [22–25], while social and psychological mechanisms, such as the ones that lead individuals to mark their ethnic identity [26] or conformism [27], will also tend to act against rapid change in an individual’s behavior. Thus, given these forces that can act against cultural change, one can ask what is the characteristic pace of cultural evolution, and how does it compare to the pace of biological evolution? In this study, I try to answer Cultural evolution is expected to be faster than biological evolution because of its Lamarckian nature, and because cultural information is transmitted through different routes than genetic information. While variation in biological evolution arises from random mutations, Lamarckian-like guided variation, which occurs through modifications to knowledge, skills and technologies made by an individual that are subsequently transmitted to other individuals, is a potent source of cultural variation [3,5,7,15,16]. Thus, in contrast to biological evolution, which is blind, cultural evolution can be a directed and consequently faster process. The pace of biological evolution is also constrained by the generation time of the species, since genetic information is transmitted vertically through sexual reproduction. While cultural information September 2012 \| Volume 7 \| Issue 9 \| e45150 1 PLOS ONE \| www.plosone.org Pace of Cultural Evolution The distributions of biological and cultural rates are significantly different (two-samples Kolmogorov-Smirnov Z = 7.044; two-tailed P,0.001). Archaeological rates are on average faster than biological rates, with the fastest cultural rates observed being more than twice as large as the fastest biological rates (Fig. 1). these questions by comparing the rates of change in technologies, as observed in the historical and archaeological record to the rates of morphological change, as seen in contemporary and fossil animal populations. d~(lnx2{lnx1)=Dt d~(lnx2{lnx1)=Dt where x1 and x2 are the mean trait value at time 1 and time 2, respectively, and Dt is the time interval between x1 and x2, measured in millions of years. The darwin is preferred to the haldane [31], which is another metric of the rate of evolutionary change, since standard deviations for metric attributes are often not reported in the archaeological literature and because the generation time for cultural evolution is unclear. The rates analyzed in this study were absolute and non-autonomous [28,32]. Materials and Methods Effort was made to assemble an unbiased sample of cultural rates calculated from what the author(s) of each study interpreted as historically continuous populations. Both biological and cultural rates were measured in darwins (d), which is a standardized unit of change in factors of e, the base of the natural logarithm, per millions of years. Results At a larger time interval of 1000 years, which is the time scale of millennial climatic fluctuations [41,47–52], the divergence is more pronounced, with morphologies changing at a pace of 60.85 d(x2/x1 = 1.062) compared to 348.505 d(x2/x1 = 1.4169) for technologies. Thus, Comparing cultural rates of change to biological rates that are calculated from various organisms on an absolute time scale allows us to compare the ability of human culture and biological traits to keep pace with a common driver, such as climate change. Historical contingencies can influence evolution [33–35], and both biological and cultural traits can be subjected to a wide range of evolutionary forces, including neutral drift and selection [8]. Identifying the precise evolutionary forces acting on biological and cultural traits, and, for instance, analyzing ‘functional’ traits separately from ‘neutral’ traits, is not a trivial task [36,37]. In order to circumvent this problem and to obtain the typical pace of biological and cultural evolution, I compare large samples of rates representing a wide range of biological and cultural traits that have undoubtedly been subjected to different regimes of evolutionary forces and historical contingencies. September 2012 \| Volume 7 \| Issue 9 \| e45150 2 PLOS ONE \| www.plosone.org Pace of Cultural Evolution Table 1. Source of the cultural rates from the archaeological record (dataset available upon request.). Results Technological trait N of Rates Absolute rate (d) American Bottom Woodland lithic point maximum length [57] 36 110–21895 American Bottom Woodland lithic point maximum width [57] 77 10–74,901 American Bottom Woodland lithic point stem length [57] 36 697–42,713 American Southwest mano length [58] 6 160–12,348 Anasazi pit structure depth [59] 6 1,304–3,529 Annapolis printer type block height [60] 26 7–3,957 Chesapeake pipe stems diameter [61] 3 252–2,424 Colorado fire features diameter [62] 15 44–3,982 Dagger blade length [63] 6 398–9,956 Dagger blade thickness [63] 3 613–1,842 Dagger blade width [63] 6 201–5,753 Delaware lithic projectile point width [64] 11 217–1,596 European farmhouses length [65] 5 959–1,438 Great point metal projectile point length [66] 3 371–12,730 Knife River Indian Villages glass bead size [67] 19 1,263–14,932 Longhouse length [68] 1 4,684 Michigan and Ontario lithic bifaces base width [69] 15 143–1,079 Missouri ceramic vessel thickness [70] 3 62–490 Missouri ceramic wall thickness [71] 16 58–17,153 Missouri Woodland vessel wall thickness [72] 44 86–12,603 Neutral lithic projectile point length [73] 25 420–45,554 Neutral lithic projectile point width [73] 15 118–37,898 New England clay pipe stems diameter [74] 15 3,364–6,372 New York state Iroquoian ceramic vessel thickness [75] 77 28–4,957 Ontario Iroquois longhouse length [76] 6 309–3,659 Pacific Northwest window glass thickness [77] 25 45,53–29,580 Portuguese ceramic vessel rim diameter [78] 1 1,482 Saskatchewan brass, projectile point length [79] 3 1,820–18,950 Saskatchewan iron and steel projectile point length [79] 28 1,161–614,969 Shoshone River valley projectile points perimeter [80] 36 8.92–1,600 Upland Mogollon pithouse depth [81] 4 943–3,187 Upper Mississippi valley ceramic vessel neck diameter [82] 6 572–3,202 doi:10.1371/journal.pone.0045150.t001 doi:10.1371/journal.pone.0045150.t001 cloud of biological rates plotted in Figure 2, actually increases both the distance between biological and cultural rates as well as the rate at which this difference increases with the time interval (Table 4). cultural evolution is faster than biological evolution when the effects of observational time intervals are controlled. This is due to the fact that for any given time interval, the characteristic amount of accumulated changes (x2 – x1) is greater for culture than for biology. This suggests that cultural change, like biological change, is a multiplicative process: the increments of change in a trait increase as the trait becomes larger. The observation that technologies can change over a short time scale, even if unnoticeably faster than animal morphologies, may be sufficient to make culture adaptive. September 2012 \| Volume 7 \| Issue 9 \| e45150 Results The minimal time interval at which biological evolution can be observed depends on the generation time of the species. For instance, biological rates calculated over a very short time interval, as plotted in Figure 2, come from species where the life span is counted in months. Several cultural rates have been observed over similarly short time scales, while the generation time of our species is approximately 20 years. Another way to look at this is to calculate evolutionary rates as change per generation, rather than as change per million years. To do this, I have assumed a generation length for humans of 20 years for the computation of cultural rates. The biological rates calculated from the fossil record were also excluded from the analysis, because we do not have good generation time estimates More surprising, however, is the fact that the magnitude of accumulated cultural changes grows at an increasingly faster pace with time, which is more so than the magnitude of accumulated biological change. Figure 2 shows that the difference between rates of cultural evolution and biological evolution may appear small or even non-existent over a short time scale. However, given enough time, cultural evolution will lead to increasingly larger ‘‘morpho- logical’’ changes than biological evolution, potentially allowing for better accommodation to selective challenges. This result is not an artifact of the bimodal distribution of the time intervals of biological rates. Removing the rates obtained from the paleonto- logical record from the analysis, which account for the rightmost September 2012 \| Volume 7 \| Issue 9 \| e45150 PLOS ONE \| www.plosone.org 3 Pace of Cultural Evolution Figure 1. Box-and-whisker plot of the distribution of the absolute values of rates on a natural log scale. On a linear scale, the distribution of biological rates (n = 503) has a mean and standard deviation of 3,187614,457d, respectively, and a range of 0.003–298,103.5d. The distribution of cultural rates (n = 573) has a mean of 4,709627,069d and a range of 7–614,969d. doi:10.1371/journal.pone.0045150.g001 Figure 1. Box-and-whisker plot of the distribution of the absolute values of rates on a natural log scale. On a linear scale, the distribution of biological rates (n = 503) has a mean and standard deviation of 3,187614,457d, respectively, and a range of 0.003–298,103.5d. The distribution of cultural rates (n = 573) has a mean of 4,709627,069d and a range of 7–614,969d. doi:10.1371/journal.pone.0045150.g001 Figure 2. Results It is possible that some of the biological rates calculated over a short time interval reflect rapid phenotypic change, while the changes observed over longer time intervals are mostly due to genetic change; and (5) the amount of cultural change observed per generation time (20 years) is significantly faster than what we would expect from biological evolution for a species with the same generation time as humans. This observation highlights the fact that culture allows us to evolve over time scales that are normally accessible only to short-lived species, while at the same time allowing us to enjoy the benefits of having a long life history, such as a large brain, an extended juvenile period, and long life span. for them. This analysis shows that the pace of cultural change, per generation, is faster than the rate of biological evolution over all observation time intervals (Fig. 4, Table 5), including time intervals that are equal or shorter than the generation time of humans. Results Biological rates and cultural rates plotted against the interval of time over which the rates are measured. Biological rates (black circles) and cultural rates (gray squares) are inversely correlated to the interval of the time over which the rates are measured. The solid lines represent the linear mixed model of interaction that best fit the biological (black line) and cultural (gray line) distributions (Table 2). doi:10.1371/journal.pone.0045150.g002 Figure 2. Biological rates and cultural rates plotted against the interval of time over which the rates are measured. Biological rates (black circles) and cultural rates (gray squares) are inversely correlated to the interval of the time over which the rates are measured. The solid lines represent the linear mixed model of interaction that best fit the biological (black line) and cultural (gray line) distributions (Table 2). doi:10.1371/journal.pone.0045150.g002 September 2012 \| Volume 7 \| Issue 9 \| e45150 PLOS ONE \| www.plosone.org 4 Pace of Cultural Evolution Table 2. Estimates of fixed effects and covariance parameters for the linear mixed model of interaction between rate, type of rate (biological or cultural rates), and time interval. Parameter Estimate 6 S.E.M Type of rate1 23.0886.455 (P,0.001) Ln (Time Interval) 20. 5996.01 (P,0.001) TypeLn (Time Interval) 20.1946.045 (P,0.001) Intercept 1.716.45 (P,0.001) Residual .91660.051 Variance random intercept .95260.121 1Cultural rates = 0; Biological rates = 1; The test is performed on the logged (ln) values of rates and time intervals. Number of biological rates = 503, number of cultural rates = 573. doi:10.1371/journal.pone.0045150.t002 Table 3. Estimates of fixed effects and covariance parameters for the linear mixed model describing the correlation between rates of cultural change as a function of their age, expressed in years BP. Parameter Estimate 6 S.E.M Ln (Years BP) 2.25276.1 (P = 0.021) Intercept 8.766.1 (P = 0.021) Residual 1.1960.072 Random Intercept Variance 1.0360.298 The test is performed on the logged (ln) values of rates and ages. Number of biological rates = 503, number of cultural rates = 573. doi:10.1371/journal.pone.0045150.t003 Table 2. Estimates of fixed effects and covariance parameters for the linear mixed model of interaction between rate, type of rate (biological or cultural rates), and time interval. Table 3. Estimates of fixed effects and covariance parameters for the linear mixed model describing the correlation between rates of cultural change as a function of their age, expressed in years BP. Results Parameter Estimate 6 S.E.M Ln (Years BP) 2.25276.1 (P = 0.021) Intercept 8.766.1 (P = 0.021) Residual 1.1960.072 Random Intercept Variance 1.0360.298 The test is performed on the logged (ln) values of rates and ages. Number of biological rates = 503, number of cultural rates = 573. doi:10.1371/journal.pone.0045150.t003 The test is performed on the logged (ln) values of rates and ages. Number of biological rates = 503, number of cultural rates = 573. doi:10.1371/journal.pone.0045150.t003 1Cultural rates = 0; Biological rates = 1; The test is performed on the logged (ln) values of rates and time intervals. Number of biological rates = 503, number of cultural rates = 573. doi:10.1371/journal.pone.0045150.t002 change. This could mean that some of the adaptive benefits of cultural transmission are not reaped during the short term, but rather occur over longer time scales. However, the fact that biological rates decrease more rapidly with time interval could be driven in part by individual phenotypic plasticity. Phenotypic plasticity is comparable to cultural evolution and allows organisms to change over short time intervals. It is possible that some of the biological rates calculated over a short time interval reflect rapid phenotypic change, while the changes observed over longer time intervals are mostly due to genetic change; and (5) the amount of cultural change observed per generation time (20 years) is significantly faster than what we would expect from biological evolution for a species with the same generation time as humans. This observation highlights the fact that culture allows us to evolve over time scales that are normally accessible only to short-lived species, while at the same time allowing us to enjoy the benefits of having a long life history, such as a large brain, an extended juvenile period, and long life span. change. This could mean that some of the adaptive benefits of cultural transmission are not reaped during the short term, but rather occur over longer time scales. However, the fact that biological rates decrease more rapidly with time interval could be driven in part by individual phenotypic plasticity. Phenotypic plasticity is comparable to cultural evolution and allows organisms to change over short time intervals. doi:10.1371/journal.pone.0045150.t005 doi:10.1371/journal.pone.0045150.t005 populations. Large and rapid changes can obscure phylogenetic relationships and lead to a loss of visibility of the most rapid changes from the data [28]. Fossil species are more likely to retain several homologous traits over longer periods of time compared to culturally transmitted technologies. This may allow for fossils to be compared despite a rapid evolution of other traits. The retention of trait homologies also allows fossils to be compared over greater spatial distances, which increases the chance that they have experienced divergent selective pressure and subsequently a greater morphological differentiation. In contrast, it is often impossible to distinguish rapid cultural evolution in the archaeological record from population replacement. Because of this uncertainty, the cultural rates that were analyzed in this study originated from populations that remained in the same locality, with spatial propinquity being used as a measurement of historical continuity. Instances of rapid cultural evolution over short time scales may therefore be underrepresented in these samples. Finally, an underlying assumption in this study is that the pace of the change of technologies is as representative of cultural evolution, in general, as the pace of morphological change is representative of biological evolution, which makes the two comparable. Yet, it is unclear as to what extent these findings Figure 4. Biological rates and cultural rates calculated as change per generation. Biological rates (black circles) and cultural rates (gray squares), calculated as change per generation, are inversely correlated to the interval of the time over which the rates are measured. The solid lines represent the linear mixed model of interaction that best fit the biological (black line) and cultural (gray line) distributions (Table 5). The conversion of rates from an absolute time scale to a generational time scale impacts mostly the biological rates from species with a short generation time. For any given amount of phenotypic change observed over any given time interval, the difference between rate of change per millions of years and rates of change per generation time increases as generation time of species increases. This effect explains why the difference between the slopes of the two linear models, although significant, is smaller (Table 5) than that shown in Figure 2. doi:10.1371/journal.pone.0045150.g004 Figure 4. Biological rates and cultural rates calculated as change per generation. Discussion In this study, I aimed to test the hypothesis that the pace of cultural evolution is faster than the pace of biological evolution. I found that (1) Similar to biological rates, rates of cultural change are inversely correlated with the time interval over which they are measured; (2) this inverse correlation explains, at least in part, why the pace of change appears to be faster for more recent periods of the archaeological record; (3) when controlling for this inverse correlation, rates of cultural evolution are significantly faster than rates of biological evolution; (4) The magnitude of accumulated cultural change grows at an increasingly faster rate with time, which is greater than the magnitude of accumulated biological However, it is likely that the archaeological record under- estimates rates of cultural change over small time intervals compared to the fossil record. Correct measurements of evolu- tionary rates must be calculated using phylogenetically-linked Figure 3. Rates of cultural evolution are inversely correlated with their age. The age of a rate corresponds to the midpoint, in years Before Present (BP), of the time interval over which the rate is calculated. The solid line represents the linear mixed model of interaction of rates with age (Table 3). doi:10.1371/journal.pone.0045150.g003 Figure 3. Rates of cultural evolution are inversely correlated with their age. The age of a rate corresponds to the midpoint, in years Before Present (BP), of the time interval over which the rate is calculated. The solid line represents the linear mixed model of interaction of rates with age (Table 3). doi:10.1371/journal.pone.0045150.g003 doi:10.1371/journal.pone.0045150.g003 September 2012 \| Volume 7 \| Issue 9 \| e45150 PLOS ONE \| www.plosone.org 5 Pace of Cultural Evolution Table 4. Estimates of fixed effects and covariance parameters for the linear mixed model of interaction between rate, type of rate (biological or cultural rates), and time interval. Parameter Estimate 6 S.E.M Type of rate1 23.9861.14 (P = 0.001) Ln (Time Interval) 2.66.04 (P,0.001) TypeLn (Time Interval) 2.286.1 (P = 0.008) Intercept 1.76.46 (P,0.001) Residual .92860.054 Variance random intercept 1.2960.187 1Cultural rates = 0; Biological rates = 1; The test is performed on the logged (ln) values of rates and time intervals. Rates calculated from the fossil record are excluded from the analysis. Number of biological rates = 283, number of cultural rates = 573. doi:10.1371/journal.pone.0045150.t004 Table 5. Controlling for the effect of generation time. Discussion Parameter Estimate 6 S.E.M Type of rate1 23.916.02 (P = 0.01) Ln (Time Interval) 2.5996.05 (P,0.001) TypeLn (Time Interval) 2.02660.15 (P,0.001) Intercept 29.126.55 (P,0.001) Residual .92760.05 Variance random intercept 3.8860.43 1Cultural rates = 0; Biological rates = 1; Estimates of fixed effects and covariance parameters for the linear mixed model of interaction between rate, type of rate (biological or cultural rates), and time interval. The analysis controls for generation time by calculating rates as the amount of change per generation time (cultural rates are calculated assuming a generation time of 20 years). The test is performed on the logged (ln) values of rates and time intervals. Number of biological rates = 283, number of cultural rates = 573. d i 10 1371/j l 0045150 t005 Table 5. Controlling for the effect of generation time. Table 4. Estimates of fixed effects and covariance parameters for the linear mixed model of interaction between rate, type of rate (biological or cultural rates), and time interval. Parameter Estimate 6 S.E.M Type of rate1 23.916.02 (P = 0.01) Ln (Time Interval) 2.5996.05 (P,0.001) TypeLn (Time Interval) 2.02660.15 (P,0.001) Intercept 29.126.55 (P,0.001) Residual .92760.05 Variance random intercept 3.8860.43 Parameter Estimate 6 S.E.M Type of rate1 23.916.02 (P = 0.01) Ln (Time Interval) 2.5996.05 (P,0.001) TypeLn (Time Interval) 2.02660.15 (P,0.001) Intercept 29.126.55 (P,0.001) Residual .92760.05 Variance random intercept 3.8860.43 Parameter Estimate 6 S.E.M Type of rate1 23.9861.14 (P = 0.001) Ln (Time Interval) 2.66.04 (P,0.001) TypeLn (Time Interval) 2.286.1 (P = 0.008) Intercept 1.76.46 (P,0.001) Residual .92860.054 Variance random intercept 1.2960.187 1Cultural rates = 0; Biological rates = 1; The test is performed on the logged (ln) values of rates and time intervals. Rates calculated from the fossil record are excluded from the analysis. Number of biological rates = 283, number of cultural rates = 573. doi:10.1371/journal.pone.0045150.t004 1Cultural rates = 0; Biological rates = 1; Estimates of fixed effects and covariance parameters for the linear mixed model of interaction between rate, type of rate (biological or cultural rates), and time interval. The analysis controls for generation time by calculating rates as the amount of change per generation time (cultural rates are calculated assuming a generation time of 20 years). The test is performed on the logged (ln) values of rates and time intervals. Number of biological rates = 283, number of cultural rates = 573. Discussion 1Cultural rates = 0; Biological rates = 1; The test is performed on the logged (ln) values of rates and time intervals. Rates calculated from the fossil record are excluded from the analysis. Number of biological rates = 283, number of cultural rates = 573. doi:10.1371/journal.pone.0045150.t004 References Contemporary Microevolution: Rate, Pattern, and Process. Dordrecht: Kluwer Academic Publishers. 127–144. 9. Boyd R, Richerson PJ (1996) Why culture is common, but cultural evolution is rare. In: Runciman WG, J MS, Dunbar RIM, editors. Proceedings of the British Academy of Science. Oxford, UK: Oxford University Press. 77–93. 9. Boyd R, Richerson PJ (1996) Why culture is common, but cultural evolution is rare. In: Runciman WG, J MS, Dunbar RIM, editors. Proceedings of the British 39. Bookstein FL (1987) Random walk and the existence of evolutionary rates. Paleobiology 13: 446–464. 10. Dean LG, Kendal RL, Schapiro SJ, Thierry B, Laland KN (2012) Identification of the Social and Cognitive Processes Underlying Human Cumulative Culture. Science 335: 1114–1118. 40. 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Richerson, Sarah Mathew, and the anonymous reviewers for their useful comments and criticisms on the various versions of the paper. doi:10.1371/journal.pone.0045150.t005 Biological rates (black circles) and cultural rates (gray squares), calculated as change per generation, are inversely correlated to the interval of the time over which the rates are measured. The solid lines represent the linear mixed model of interaction that best fit the biological (black line) and cultural (gray line) distributions (Table 5). The conversion of rates from an absolute time scale to a generational time scale impacts mostly the biological rates from species with a short generation time. For any given amount of phenotypic change observed over any given time interval, the difference between rate of change per millions of years and rates of change per generation time increases as generation time of species increases. This effect explains why the difference between the slopes of the two linear models, although significant, is smaller (Table 5) than that shown in Figure 2. doi:10.1371/journal.pone.0045150.g004 Figure 4. Biological rates and cultural rates calculated as change per generation. Biological rates (black circles) and cultural rates (gray squares), calculated as change per generation, are inversely correlated to the interval of the time over which the rates are measured. The solid lines represent the linear mixed model of interaction that best fit the biological (black line) and cultural (gray line) distributions (Table 5). The conversion of rates from an absolute time scale to a generational time scale impacts mostly the biological rates from species with a short generation time. For any given amount of phenotypic change observed over any given time interval, the difference between rate of change per millions of years and rates of change per generation time increases as generation time of species increases. This effect explains why the difference between the slopes of the two linear models, although significant, is smaller (Table 5) than that shown in Figure 2. doi:10.1371/journal.pone.0045150.g004 September 2012 \| Volume 7 \| Issue 9 \| e45150 PLOS ONE \| www.plosone.org 6 Pace of Cultural Evolution can be extended to other domains of human cultures, such as social norms, institutions, or political structures, or compared to known rates of change in language [53–55] and performance of modern information technologies [56]. While many questions about the pace of cultural evolution remain to be resolved, the study presented here illustrates the utility of an archaeological perspective on patterns of cultural evolution. 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https://openalex.org/W4299862887	https://zenodo.org/records/6803597/files/OWCAtlas_Mayfly.pdf	English	null	Atlas of the Mayfly Larvae (Class Insecta: Order Ephemeroptera) Recorded at the Old Woman Creek National Estuarine Research Reserve & State Nature Preserve, Ohio	Zenodo (CERN European Organization for Nuclear Research)	2,007	cc-by	3,158	Atlas of the Mayfly Larvae (Class Insecta: Order Ephemeroptera) Recorded at the Old Woman Creek National Estuarine Research Reserve & State Nature Preserve, Ohio Atlas of the Mayfly Larvae (Class Insecta: Order Ephemeroptera) Recorded at the Old Woman Creek National Estuarine Research Reserve & State Nature Preserve, Ohio Acknowledgments The authors are grateful for the assistance of Dr. David Klarer, Old Woman Creek National Estuarine Research Reserve, for providing funding for this project and for his critical reviews of drafts. This work was funded under contract to Heidelberg College by the Ohio Department of Natural Resources, Division of Natural Areas and Preserves. “This publication was supported [in part] by Grant Number H50/CCH524266 from the Centers for Disease Control and Prevention. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of Centers for Disease Control and Prevention.” from the Centers for Disease Control and Prevention. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of Centers for Disease Control and Prevention.” “The Old Woman Creek National Estuarine Research Reserve in Ohio is part of the National Estuarine Research Reserve System (NERRS), established by Section 315 of the Coastal Zone Management Act, as amended. Additional information about the system can be obtained from the Estuarine Reserves Division, Office of Ocean and Coastal Resource Management, National Oceanic and Atmospheric Administration, U.S. Department of Commerce, 1305 East West Highway – N/ORM5, Silver Spring, MD 20910. Financial support for this publication was provided by a grant under the Federal Coastal Zone Management Act, administered by the Office of Ocean and Coastal Resource Management, National Oceanic and Atmospheric Administration, Silver Spring, MD.” Copies of this publication are available from the Ohio Department of Natural Resources-Division of Wildlife 2514 Cleveland Road East Huron, Ohio 44839 “The Old Woman Creek National Estuarine Research Reserve in Ohio is part of the National Estuarine Research Reserve System (NERRS), established by Section 315 of the Coastal Zone Management Act, as amended. Additional information about the system can be obtained from the Estuarine Reserves Division, Office of Ocean and Coastal Resource Management, National Oceanic and Atmospheric Administration, U.S. Department of Commerce, 1305 East West Highway – N/ORM5, Silver Spring, MD 20910. Financial support for this publication was provided by a grant under the Federal Coastal Zone Management Act, administered by the Office of Ocean and Coastal Resource Management, National Oceanic and Atmospheric Administration, Silver Spring, MD.” Copies of this publication are available from the Ohio Department of Natural Resources-Division of Wildlife 2514 Cleveland Road East Huron, Ohio 44839 Introduction Female imago (adult) of Hexagenia sp., length (excluding tail filaments) about 3 cm (1.25 inches). Acknowledgments Shed skin of subimago is partly visible at lower left. Both the formal biologist and the amateur naturalist often encounter lists of animals and plants when they read published scientific reports and visit nature centers. Rarely do they have ready access to photographs for each member of the list. The purpose of this chapter of the atlas is to provide a detailed pictorial record of the larval mayflies (Class Insecta: Order Ephemeroptera) within the Old Woman Creek coastal wetland system (OWC) along Lake Erie in Ohio. Female imago (adult) of Hexagenia sp., length (excluding tail filaments) about 3 cm (1.25 inches). Shed skin of subimago is partly visible at lower left. As a group, mayfly larvae live in a wide variety of aquatic habitats. Some kinds live in both standing and running water; other kinds live in only one or the other. Both their presence and abundance are excellent indicators of the quality, or “health”, of aquatic environments. Winged, nonbiting mayfly subadults (“duns” or subimagos) and adults (“spinners” or imagos) often congregate in large swarms near the shores of lakes and streams. Along the shore of Lake Erie, so many mayflies have sometimes accumulated under lights that dump trucks were required to remove them. Invertebrates occur in great abundance in freshwater ecosystems, including Great Lakes wetlands such as the marshes, swamps, and open water that make up OWC. A few of those invertebrates, such as freshwater mussels, grass shrimps, and giant water bugs, are readily visible once collected because of their large size. However, many others are so small that special attention must be given to seeing them. Most groups of invertebrates go unnoticed by casual visitors to aquatic habitats, and this is true of the aquatic larvae, or nymphs, of many kinds of mayflies. They range in length from less than 1 millimeter (1/16 inch) to more than 3 centimeters (1-1/4 inch), depending on kind and larval stage (instar). Invertebrates occur in great abundance in freshwater ecosystems, including Great Lakes wetlands such as the marshes, swamps, and open water that make up OWC. A few of those invertebrates, such as freshwater mussels, grass shrimps, and giant water bugs, are readily visible once collected because of their large size. However, many others are so small that special attention must be given to seeing them. Acknowledgments Most groups of invertebrates go unnoticed by casual visitors to aquatic habitats, and this is true of the aquatic larvae, or nymphs, of many kinds of mayflies. They range in length from less than 1 millimeter (1/16 inch) to more than 3 centimeters (1-1/4 inch), depending on kind and larval stage (instar). Winged, nonbiting mayfly subadults (“duns” or subimagos) and adults (“spinners” or imagos) often congregate in large swarms near the shores of lakes and streams. Along the shore of Lake Erie, so many mayflies have sometimes accumulated under lights that dump trucks were required to remove them. Mayflies fill a major role in aquatic ecosystems as a food source for fishes, other vertebrates, and predaceous invertebrates. Mayfly larvae generally feed by scraping or collecting their food, which consists primarily of detritus (remains of plants and animals), algae, and microorganisms. Adult mayflies do not eat or bite, as they lack functional mouthparts. Females deposit eggs at the water surface within minutes after mating. The ability to distinguish one kind of mayfly larva from another often requires careful observation through a dissecting microscope. This chapter presents detailed photographs of critical diagnostic features that permit correct identification to the taxonomic level of genus (plural, genera) of the mayfly larvae found to date at OWC. Leptophlebia sp., dorsal views G LS T A H TF WP Characteristics of Ephemeroptera Larvae T A H TF WP As members of the Order Ephemerop- tera, mayflies have four life stages: egg, larva, subimago, and imago (adult). The female lays eggs in the water, and most species of larvae hatch from a week to several months later. All larvae are aquatic and undergo more instars (periods between molts) than any other insect order. Larvae spend from 3 months to 2 or more years in the water before emerging from the water as subimagos. Mayflies are the only insects with a subimago instar, which possesses fully functional wings along with an immature reproductive system. Within approximately 24 hours, the subimago sheds its skin (molts) and becomes an imago. A Leptophlebia sp., dorsal views G LS G LS G Leptophlebia sp., dorsal views m Hexagenia sp. head, lateral view MT An General features of a mayfly larva are shown in the photographs on this page. The body is divided into three regions: head (H), thorax (T) and abdomen (A). The head of some genera displays a pair of mandibular tusks (MT) arising below (ventral to) the antennae (An). Gills (G) are attached to the sides of the abdomen (A), which may feature lateral spines (LS). Wing pads (WP) are present dorsally on the thorax of mature individuals but are absent on young larvae. Most mayfly larvae have three tail filaments (TF) attached to the end of the abdomen, although a few species have only two. Each leg is divided into five parts: the coxa (attached to the thorax, not shown), the trochanter (not shown), the femur (F), the tibia (Ti), and the tarsus (Ta). A claw (C) is attached to each tarsus. Hexagenia sp. head, lateral view Hexagenia sp. head, lateral view Hexagenia sp. hind leg, dorsal view C F Ti Ta Hexagenia sp. hind leg, dorsal view Hexagenia sp. hind leg, dorsal view Layout of this Atlas Beneath the descriptive features, each page lists where within OWC the genus has been found. It is likely that future collections will reveal some of the genera in additional habitats. The general ecology of the genus is briefly summarized, including its habit (such as swimmer or climber), its functional feeding group (such as collector- gatherer), and for some genera, special notes about its habitat or water quality requirements. The following pages are organized alphabetically by family. The authors and other collectors have identified five genera of mayflies in four families (Baetidae, Caenidae, Ephemeridae, and Leptophlebiidae) within the OWC wetland system, which excludes the free-flowing upland reaches of Old Woman Creek. This publication should not be used as the sole source to identify the families and genera of larval mayflies of OWC because it is likely that additional families and genera will be found in new collections. The references cited on this page should be used to obtain definitive identifications. The species within each genus are not included here, and the species of some genera cannot be identified. All information on each page was derived from three references, which are abbreviated as shown below followed by the page number(s): E&W = Edmunds, Jr., G.F., and R.D. Waltz. 1996. Chapter 11. Ephemeroptera, pp. 126-163. In: Merritt, R.W., and K.W. Cummins (Eds.). An Introduction to the Aquatic Insects of North America. 3rd Ed. Kendall/Hunt Publishing Co., Dubuque, Iowa. E&W = Edmunds, Jr., G.F., and R.D. Waltz. 1996. Chapter 11. Ephemeroptera, pp. 126-163. In: Merritt, R.W., and K.W. Cummins (Eds.). An Introduction to the Aquatic Insects of North America. 3rd Ed. Kendall/Hunt Publishing Co., Dubuque, Iowa. Each genus of mayfly larva is illustrated and described on a single page of this atlas. Because the identifying features of the Order Ephemeroptera and the particular family are repeated on each page, the page for each genus can be used independently. Photographs are labeled with identifying letters and lines that indicate diagnostic structures. Each genus of mayfly larva is illustrated and described on a single page of this atlas. Because the identifying features of the Order Ephemeroptera and the particular family are repeated on each page, the page for each genus can be used independently. Photographs are labeled with identifying letters and lines that indicate diagnostic structures. Some photographs show specimens collected within OWC; specimens from other ecosystems were used if they were of superior quality. Layout of this Atlas The exact specimens photographed are recorded at the bottom of the page. H = Hilsenhoff, W.L. 1982. Aquatic Insects of Wisconsin. Publication of the Natural History Council, University of Wisconsin-Madison, No. 2. Geological & Natural History Survey, Madison, Wisconsin. H = Hilsenhoff, W.L. 1982. Aquatic Insects of Wisconsin. Publication of the Natural History Council, University of Wisconsin-Madison, No. 2. Geological & Natural History Survey, Madison, Wisconsin. Some photographs show specimens collected within OWC; specimens from other ecosystems were used if they were of superior quality. The exact specimens photographed are recorded at the bottom of the page. V = Voshell, Jr., J.R. 2002. A Guide to Common Freshwater Invertebrates of North America. The McDonald and Woodward Publishing Co., Blacksburg, VA. Features of Order Ephemeroptera Three pairs of segmented legs on thorax Wing pads developing or absent Two or three terminal filaments on abdomen Plate-like, leaf-like, or feather-like gills on sides of abdomen E Insecta: Ephemeroptera: Baetidae: Baetis sp. Small Minnow Mayflies I I’ Dorsal views of Baetis Ventral view of Baetis Lateral views of Baetis G A B D C 1 mm J H E F 9 8 2 3 I’ Dorsal views of Baetis A E F 9 8 2 3 Features of Family Baetidae Abdominal gills consisting of one or two processes, exposed and present on abdominal segments 1 or 2 through 7; gills on abdominal segment 2 similar to those on succeeding segments (A) Mandibular tusks absent (B) Long, dense hair on forelegs absent (C) Head not flattened dorso-ventrally (D) Eyes and antennae lateral (not dorsal) (E) Distinct lateral spines absent on abdominal segments 8 and 9 (F) Features of Genus Baetis Three terminal filaments present (G) Gills present as single processes (A,G,H) Claws on tarsus stout (I), shorter than half the length of tarsus (I’) Large teeth on tarsal claws (J) F I I’ J F C Where Recorded at Old Woman Creek Standing water in swamp forest General Ecology Habit: Swimmers, climbers, clingers Functional feeding group: Scrapers, Collectors-gatherers of detritus and diatoms References: E&W 132-135, 159, 162; H 2, 9-12; V 144 Photographs: St. Johns Dam MEX August 27, 2004; St. Johns Dam CH 90 August 19, 2004 Lateral views of Baetis B D H Ventral view of Baetis G 1 mm Ventral view of Baetis Lateral views of Baetis B D H H Insecta: Ephemeroptera: Baetidae: Callibaetis sp. Small Minnow Mayflies 1 mm Small Minno Dorsal view of Callibaetis Ventral views of Callibaetis Lateral views of Callibaetis C G A 2 10 9 8 7 6 5 4 3 D B I H E 1 mm Dorsal view of Callibaetis H E Features of Order Ephemeroptera Three pairs of segmented legs on thorax Wing pads developing or absent Two or three terminal filaments on abdomen Plate-like, leaf-like, or feather-like gills on sides of abdomen Features of Family Baetidae Abdominal gills consisting of one or two processes, exposed and present on abdominal segments 1 or 2 through 7; gills on abdominal segment 2 similar to those on succeeding segments (A) Mandibular tusks absent (B) Long, dense hair on forelegs absent (C) Head not flattened dorso-ventrally (D) Eyes and antennae lateral (not dorsal) (E) Distinct lateral spines absent on abdominal segments 8 and 9 (F) Lateral views of Callibaetis Ventral views f C llib i C G A 2 10 9 8 7 6 5 4 3 D B I 2 3 D B I D B D D Features of Genus Callibaetis Claws slender with sharp points (G) Three terminal filaments present (H) Gills with distinct ventral flaps (I) C G A 10 9 8 7 6 5 4 3 C G Where Recorded at Old Woman Creek Swamp forest; on filamentous algae General Ecology Habit: Swimmers, clingers Functional feeding group: Collectors- gatherers of filamentous algae References: E&W 132-135, 159; H 2, 9-12; V 144 Photographs: KK REF E3 July 31, 2002 OWCI; WQL REF BB3 Ventral views of Callibaetis F 9 8 1 mm F F F Insecta: Ephemeroptera: Caenidae: Caenis sp. Small Squaregill Mayflies Small Squar Dorsal views of Caenis Ventral view of Caenis Lateral view of Caenis A D C C 1 mm 1 mm B B’ Features of Order Ephemeroptera Dorsal views of Caenis C 1 mm B B’ Three pairs of segmented legs on thorax Wing pads developing or absent Two or three terminal filaments on abdomen Plate-like, leaf-like, or feather-like gills on sides of abdomen Features of Family Caenidae Mandibular tusks absent (A) Gills on abdominal segment 2 fringed with long setae on outer margin (B) and meeting or overlapping on dorsal surface of abdomen (B’), nearly square and covering the gills on segments 3-6 Gills on abdominal segments 3-6 fringed at margins (C) C C D Features of Genus Caenis No tubercles on head (D) Where Recorded at Old Woman Creek In sediment at the following locations: lotus bed, near water lily bed, drowned OWC channel at upper end of wetland, upstream creek bed General Ecology Habit: Swimmers, climbers Functional feeding group: Collectors- gatherers, scrapers References: E&W 130, 147, 162; H 2, 9; V 140 Photographs: REF E2 August 6, 2002 OWCI Features of Genus Caenis No tubercles on head (D) Ventral view of Caenis 1 mm Ventral view of Caenis 1 mm ateral view of Caenis A A A Lateral view of Caenis Insecta: Ephemeroptera: Ephemeridae: Hexagenia sp. Common Burrower Mayflies Insecta: Ephemeroptera: Ephemeridae: Hexagenia sp. Common Burrower Mayflies Common Burr Dorsal views of Hexagenia Ventral views of Hexagenia 1 cm 1 cm Lateral views of Hexagenia G E F D C B A Dorsal views of Hexagenia 1 cm E F D C Dorsal views of Hexagenia Features of Order Ephemeroptera Features of Order Ephemeroptera Three pairs of segmented legs on thorax Wing pads developing or absent Two or three terminal filaments on abdomen Plate-like, leaf-like, or feather-like gills on sides of abdomen Features of Family Ephemeridae Mandibular tusks with upward curve (A) Gills held dorsally (B) Hind tibiae forming pronounced point at apex (C) Front tibiae flattened and widened for burrowing (D) Features of Genus Hexagenia A rounded frontal process between the eyes (E) Antennae with whorls of long setae (F) Gills on segment one forked (G) Where Recorded at Old Woman Creek Sediment of lotus bed and upstream creek channel General Ecology Habit: Burrowers Functional feeding group: Collectors- gatherers References: E&W 130-132, 147, 163; H 2, 9; V 137 Photographs: KK REF BB5 August 1, 1979 UHC 2B g E Three pairs of segmented legs on thorax Wing pads developing or absent Two or three terminal filaments on abdomen Plate-like, leaf-like, or feather-like gills on sides of abdomen 1 cm F C C D Lateral views of Hexagenia Lateral views of Hexagenia B A B B Ventral views of Hexagenia 1 cm Insecta: Ephemeroptera: Leptophlebiidae: Leptophlebia sp. Pronggilled Mayflies Insecta: Ephemeroptera: Leptophlebiidae: Leptophlebia sp. Pronggilled Mayflies Dorsal views of Leptophlebia Ventral views of Leptophlebia Oblique views of Leptophlebia B G A C G’ H F E 3 mm 3 mm Dorsal views of Leptophlebia G A G’ H F 3 mm Features of Order Ephemeroptera Dorsal views of Leptophlebia 3 mm Three pairs of segmented legs on thorax Wing pads developing or absent Two or three terminal filaments on abdomen Plate-like, leaf-like, or feather-like gills on sides of abdomen H Features of Family Leptophlebiidae Abdominal gills exposed on all segments (A) Mandibular tusks absent (B) Long, dense hair on forelegs absent (C) Gills on abdominal segment 2 similar to those on succeeding segments (A, F) Claws of all tarsi similar in structure and length (D) Abdominal gills forked (segment 1)(E), present as clustered filaments, or consisting of two pointed leaf-like plates (F) (dorsal plate obscuring ventral plate in photo) Oblique views of Leptophlebia Ventral views of Leptophlebia Ventral views of Leptophlebia Oblique views of Leptophlebia B C E 3 mm Features of Genus Leptophlebia Head (G) wider than labrum (G’) Antennae and eyes lateral (not dorsal) (H) Gills on abdominal segment 1 forked (E); succeeding gills each consist of two leaf- like plates (F) Where Recorded at Old Woman Creek Benthic in wetland, undesignated habitat General Ecology Habit: Swimmers, sprawlers, clingers Functional feeding group: Collectors- gatherers References: E&W 130-132, 139, 163; H 2, 9-12; V 143 Photographs: KK REF BB4 March 20, 1980 UHC1 D D
https://openalex.org/W2805196824	http://ira.lib.polyu.edu.hk/bitstream/10397/78681/1/Hussain_Tensile_Behavior_Cu.pdf	English	null	Tensile behavior of Cu-coated Pd40Cu30Ni10P20 metallic glassy wire	Scientific reports	2,018	cc-by	6,750	Tensile behavior of Cu-coated Pd40Cu30Ni10P20 metallic glassy wire I. Hussain1,2, Y. Y. Jiang1, Y. D. Jia1, G. Wang1, Q. J. Zhai1, K. C. Chan3 & J. Yi1 I. Hussain1,2, Y. Y. Jiang1, Y. D. Jia1, G. Wang1, Q. J. Zhai1, K. C. Chan3 & J. Yi1 Catastrophic brittle fracture of monolithic metallic glass (MG) hinders engineering application of MGs. Although many techniques has been tried to enhance tensile ductility of metallic glasses, the enhancement is quite limited. Here, we show the effect of electrodeposited Cu coating on tensile plasticity enhancement of Pd40Cu30Ni10P20 MG wires, with different volume fractions of copper coatings (R), from 0% to 97%. With increasing R, tensile elongation is enhanced to 7.1%. The plasticity enhancement is due to confinement of the Cu coatings, which lead to multiple and secondary shear bands, according to SEM investigations. In addition, the SEM images also show that the patterns on the fracture surface of the Cu-coated MG wires vary with volume fraction of the Cu coatings. The size of shear offset decreases with increasing R. The viscous fingerings on the fracture surface of monolithic MG wire changes into dimples on the fracture surface of Cu coated MG wires with R of 90% and 97%. The electrodeposition technique used in this work provides a useful way to enhance plasticity of monolithic MGs under tensile loading at room temperature. Received: 11 January 2018 Accepted: 22 March 2018 Published: xx xx xxxx Received: 11 January 2018 Accepted: 22 March 2018 Published: xx xx xxxx Plasticity of bulk and micro-sized MG is mediated by shear bands with highly localized strain1,2. However, in tension, the tensile stress enhances softening and instability, a dominant shear band slips without obstacle, and bulk and micro-sized MGs fracture elastically3. Various approaches have been introduced to retard the dominant shear band to improve tensile ductility of bulk and micro-sized MGs, such as designing MG matrix composites4,5, sharp-and-deep notches6, laminating MG with ductile crystalline metals7,8, surface mechanical treatment9–11, laser surface texturing treatment12, and MG-based chiral nanolattice13. In addition, size reduction approach changes room temperature plastic deformation mechanism of MG from shear banding into homogeneous plastic flow14–18. However, the transition occurs at submicron length scale17,19. l At the same time, other techniques have been introduced to improve compressive plasticity of bulk MGs. What attracted our attention was metal coating technique. By using this technique, compressive plasticity of Fe-based metallic glass was improved from 0.5% to 5.0%20. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Tensile behavior of Cu-coated Pd40Cu30Ni10P20 metallic glassy wire I. Hussain1,2, Y. Y. Jiang1, Y. D. Jia1, G. Wang1, Q. J. Zhai1, K. C. Chan3 & J. Yi1 Furthermore, a theoretical study has shown that a thin metal coating slows down shear band dynamics and retards its attainment to a critical unstable state21. However, metal coating effects on tensile plasticity of MGs have not yet been investigated. f Here, Pd40Cu30Ni10P20 MG wires are chosen for investigation, because such wires can have much higher ratio of lateral surface area to cross section area that provide sufficient adhesive force to prevent sliding between the MG core and the metal coating, as described in the literature21. Cu is chosen to be electrodeposited onto the Pd40Cu30Ni10P20 MG wires for investigation, because the Cu electrodeposition technique is flexible and facile. It is shown that tensile ductility of the Cu coated MG wires increases significantly with increasing Cu coating content. At the same time, fracture morphology changes from viscous fingering to dimpling. Received: 11 January 2018 Accepted: 22 March 2018 Published: xx xx xxxx Results C ti Coating quality. High quality coating is expected for high mechanical performance of metal coated mate- rials8. Therefore, before tensile tests, the coating quality was investigated using scanning electron microscope (SEM). The SEM images in Fig. 1 show surface (Fig. 1(a)) and the cross-sectional (Fig. 1(b)) of morphology a Cu-coated Pd40Cu30Ni10P20 MG wire. Figure 1(a) shows homogeneous Cu coating surface without any detected defects. At the same time, the cross-sectional image of the coated wire in Fig. 1(b) shows that the Cu layer is con- tinuous without pore; and no defects, such as voids and/or cracks, can be detected at or near the interface between the coating and the MG core. All these images confirm that high quality Cu coating was well adhered to the MG wire. The high quality electrodeposition was kept from sample to sample by using constant electrodeposition 1Laboratory for Microstructures, Institute of Materials, Shanghai University, Shanghai, 200444, China. 2Department of Chemistry, Karakoram International University, Gilgit-Baltistan, 15100, Pakistan. 3Department of Industrial and System Engineering, The Hong Kong Polytechnic University, Hong Kong, China. Correspondence and requests for materials should be addressed to K.C.C. (email: kc.chan@polyu.edu.hk) or J.Y. (email: jxy305@gmail.com) ScIentIfIc RePorTs \| (2018) 8:5659 \| DOI:10.1038/s41598-018-23956-5 1 www.nature.com/scientificreports/ Figure 1. Surface and cross-sectional morphology of Cu-coated Pd40Cu30Ni10P20 MG wires: (a) optical image of the cross section of Cu-coated Pd40Cu30Ni10P20 MG wire; (b) SEM image of the surface of homogeneous Cu coating without detected defects. Figure 1. Surface and cross-sectional morphology of Cu-coated Pd40Cu30Ni10P20 MG wires: (a) optical image of the cross section of Cu-coated Pd40Cu30Ni10P20 MG wire; (b) SEM image of the surface of homogeneous Cu coating without detected defects. Figure 2. Engineering tensile stress-strain curves of Cu-coated Pd40Cu30Ni10P20 MG wires with coating volume fraction R of 0%, 45%, 70%, 90%, and 97%, and electrodeposited pure Cu. The straight dash lines are eyesight guide of the tensile stress-strain curves of the as-cast Pd40Cu30Ni10P20 MG wire and the wire with R of 45% for showing the nonlinearity of the curve. The inset indicates five stages of tensile deformation process of Cu- coated Pd40Cu30Ni10P20 MG wires. In stage I, both the coating and the wire core deform linearly. In stage II, the coating deforms plastically while the wire core elastically. In stage III, both the coating and the wire core deform plastically. In stage IV, the wire core fractures. In stage V, the coating necks and then fractures. Figure 2. Results C ti Engineering tensile stress-strain curves of Cu-coated Pd40Cu30Ni10P20 MG wires with coating volume fraction R of 0%, 45%, 70%, 90%, and 97%, and electrodeposited pure Cu. The straight dash lines are eyesight guide of the tensile stress-strain curves of the as-cast Pd40Cu30Ni10P20 MG wire and the wire with R of 45% for showing the nonlinearity of the curve. The inset indicates five stages of tensile deformation process of Cu- coated Pd40Cu30Ni10P20 MG wires. In stage I, both the coating and the wire core deform linearly. In stage II, the coating deforms plastically while the wire core elastically. In stage III, both the coating and the wire core deform plastically. In stage IV, the wire core fractures. In stage V, the coating necks and then fractures. parameters, such as electrolyte composition, working temperature, current density, pH value, the surface state of the MG wire, electroplating time span and so forth by following previous work22. The high quality can also prevent the MG core from sliding during tension. In order to confirm this, the two ends of the sample shown in Fig. 5(b) was polished by using SiC paper along the dashed lines before tensile testing. After the tensile testing, the polished cross-sections were investigated in SEM. The SEM image in Fig. 1(c) shows no sliding can be detected along the interface between the Cu coating and the MG core. Tensile behavior of as-cast and Cu-coated MG wires. Tensile behavior of as-cast and Cu-coated Pd40Cu30Ni10P20 MG wires with specific R values of 45%, 90%, and 97% were characterized as shown in Fig. 2 by using tensile testing sample as shown in Fig. 5(b).h Tensile behavior of as-cast and Cu-coated MG wires. Tensile behavior of as-cast and Cu-coated d40Cu30Ni10P20 MG wires with specific R values of 45%, 90%, and 97% were characterized as shown in Fig. 2 by sing tensile testing sample as shown in Fig. 5(b).h g g g The stress-strain curve of as-cast wires shows that it fractures catastrophically without any plasticity as bulk metallic glasses3 and other MG wires11,23–25. The deformation of the as-cast wire shows nonlinearity before frac- ture while bulk metallic glasses do not3. The nonlinearity has been investigated both experimentally and theoret- ically23 and is outside the scope of this work.h y p The stress-strain curve of the wire with R value of 45% also shows nonlinearity. Here, the nonlinearity is mostly from plasticity of Cu coating. Results C ti Yield strength σy, tensile strength σT, strength at the fracture of MG wire σf w , , strain at the fracture of MG wire εf w , , fracture strength σf , and fracture strain εf of Cu-coated Pd40Cu30Ni10P20 MG wires with different R values. Table 1. Yield strength σy, tensile strength σT, strength at the fracture of MG wire σf w , , strain at the fracture of MG wire εf w , , fracture strength σf , and fracture strain εf of Cu-coated Pd40Cu30Ni10P20 MG wires with different R values. E of the Cu-coated Pd40Cu30Ni10P20 MG wires cannot be calculated as volume-weighted E of the coating and the MG core as that of composites reinforced by aligned continuous fiber27. Furthermore, 0.2% offset yield strength can be defined as 941 MPa as listed in Table 1. The wire with R value of 45% fracture catastrophically at a strain of 2.55% and a strength of 1199 MPa. The fracture strain is increased by 0.34% while the strength is reduced. The stress-strain curve also shows that the Cu coating and the MG core fracture simultaneously. However, this is different from compressive fracture of Cu-coated bulk metallic glass, during which the Cu coating completely peers off the metallic glass core21. In a word, the tensile deformation and fracture behavior of the Cu-coated Pd40Cu30Ni10P20 MG wires with a R value of 45% is very similar to fiber reinforced composites with high fiber volume fraction as reported in literature27. When R increases to a value of 70%, the tensile behavior of the wire changes as shown in Fig. 2. With increased R value, the yield strength σy decreases to 484 MPa as listed in Table 1. At the same time, the tensile strength σT and fracture strength σf , which are 900 MPa and 873 MPa (Table 1), respectively, do not equal to each other any- more. Therefore, strain localization happens before fracture because the tensile strength is higher than the frac- ture strength28. Furthermore, the MG core and the Cu coating of the wire still fracture concurrently as that of the wire with lower R value described above. When the R value increases to 90%, the tensile stress-strain curve in Fig. Results C ti Nevertheless, Young’s modulus E of pure Cu measured from tensile stress-strain curve is much smaller than that measured by using acoustic technique because plasticity of pure copper initiates at a very low strain value26. That’s why the slope of the linear region of stress-strain curves of wires with R value of 45% is obviously smaller than that of as-cast Pd40Cu30Ni10P20 MG wires. According to tensile stress-strain curve in literature26, the elastic deformation region and theoretical yield strength cannot be defined. Therefore, ScIentIfIc RePorTs \| (2018) 8:5659 \| DOI:10.1038/s41598-018-23956-5 2 www.nature.com/scientificreports/ Figure 3. Catastrophic shear fracture to necking transition of Cu-coated Pd40Cu30Ni10P20 MG wires. (a) Variation of the reduction of area − A A A ( )/ f 0 0 with volume fraction of Cu coating R. (b,c and d) are fracture morphology of Cu-coated Pd40Cu30Ni10P20 MG wires with R of 45%, 70% and 90%, respectively. (e) Morphology of the MG core of the Cu-coated Pd40Cu30Ni10P20 MG wires with R of 97%. Figure 3. Catastrophic shear fracture to necking transition of Cu-coated Pd40Cu30Ni10P20 MG wires. (a) Variation of the reduction of area − A A A ( )/ f 0 0 with volume fraction of Cu coating R. (b,c and d) are fracture morphology of Cu-coated Pd40Cu30Ni10P20 MG wires with R of 45%, 70% and 90%, respectively. (e) Morphology of the MG core of the Cu-coated Pd40Cu30Ni10P20 MG wires with R of 97%. R σy (MPa) σT (MPa) σf w , (MPa) εf w , (%) σf (MPa) εf (%) 0% 1633 ± 24 1633 ± 24 1633 ± 24 2.21 ± 0.322 1633 ± 24 2.21 ± 0.32 45% 941 ± 17 941 ± 17 1198 ± 21 2.49 ± 0.34 1198 ± 21 2.49 ± 0.34 70% 484 ± 16 900 ± 17 873 ± 14 2.55 ± 0.26 873 ± 15 2.55 ± 0.31 90% 371 ± 10 588 ± 13 547 ± 13 3.82 ± 0.21 260 ± 11 3.98 ± 0.20 97% 311 ± 9 498 ± 10 464 ± 9 7.09 ± 0.24 0 8.50 ± 0.18 Table 1. Yield strength σy, tensile strength σT, strength at the fracture of MG wire σf w , , strain at the fracture of MG wire εf w , , fracture strength σf , and fracture strain εf of Cu-coated Pd40Cu30Ni10P20 MG wires with different R values. Table 1. Results C ti 2 shows that deformation and fracture behavior of the wire varies a lot and is very similar to tensile stress-strain curve of aligned continuous metal fiber reinforced composites with lower fiber volume fraction27. In order to clearly describe the uniaxial stress-strain response, the curve is divided into five stages as shown in the inset of Fig. 2. In stage I, both the coating and the MG core deforms linearly. When it comes to stage II, the MG core continuously deforms elastically, because strain range of stage II is within the elastic limit of monolithic MG. At the same time, the Cu coating deforms plastically, because the relationship between stress and strain is no longer linear. In stage II, the stress decreases slightly with increasing strain, and the strain range goes outside the elastic limit of monolithic Pd40Cu30Ni10P20 MG wires. As reported in the literature29, monolithic MG undergoes softening during plastic deformation. Therefore, the plateau in the stress-strain curve indicates plastic deformation of Pd40Cu30Ni10P20 MG core. This will be further discussed in the next section. Near the end of stage III, the stress drops faster with increasing strain. Then, the stress drops abruptly and the MG core fractures in stage IV. However, the coating and the MG core do not frac- ture concurrently. Therefore, Cu coating deforms plastically and then fractures in stage V. As shown in Fig. 2, ScIentIfIc RePorTs \| (2018) 8:5659 \| DOI:10.1038/s41598-018-23956-5 3 www.nature.com/scientificreports/ www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 4. Fracture patterns on the fracture surface of Pd40Cu30Ni10P20 MG core: (a) viscous fingering on the fracture surface of as-cast Pd40Cu30Ni10P20 MG wire; (b) viscous fingerings and dimples on the fracture surface of the MG wire core of the wire with a R value of 45%; (c,d) dimples on the fracture surface of the MG wire cores in the wires with R values of 90% and 97%. Figure 4. Fracture patterns on the fracture surface of Pd40Cu30Ni10P20 MG core: (a) viscous fingering on the fracture surface of as-cast Pd40Cu30Ni10P20 MG wire; (b) viscous fingerings and dimples on the fracture surface of the MG wire core of the wire with a R value of 45%; (c,d) dimples on the fracture surface of the MG wire cores in the wires with R values of 90% and 97%. the deformation behavior of the wire with R value of 97% is very similar to that of the wire with R value of 90%. Results C ti Their difference is that the stress and strain values are different as listed in Table 1. The 0.2% offset yield strength is reduced from 371 MPa to 311 MPa. Surprisingly, the fracture strain of the Pd40Cu30Ni10P20 MG core is increased from 3.82% to 7.09% during uniaxial tensile. This indicates that metallic glassy wires can provide not only high strength but also significant ductility in fiber reinforced composites. Deformation and fracture morphology. Transition from catastrophic shear fracture to ductile frac- ture. Not only as-cast MG wire but also the wires with low R value, such as the wire with R value of 45%, fracture catastrophically as shown in Fig. 3a and b. When the R value increases to 70%, the reduction of area − A A A ( )/ f 0 0 (where A0 is the cross-sectional area and Af is the cross-sectional area of the fracture) increases to 50%, even though the ductility improvement is almost zero when it is compared with the MG wire with R value of 45%. Therefore, the transition from catastrophic shear fracture to ductile necking of the MG wires happens somewhere between R values of 45% and 70%. However, Fig. 3c shows that the final fracture still happens in shear. When the R value further increases to 90%, − A A A ( )/ f 0 0 increases to 75%, and the final fracture shown in Fig. 3d changes totally into ductile manner. The Morphology of the MG wire core electropolished from the wire with R value of 97% is shown in Fig. 3e. The image indicate that the ductility of the MG wire core is mediated by dense shear banding like compressive ductility of bulk metallic glasses30. However, no indication of necking can be found on the MG wire core. This transition from catastrophic shear fracture to ductile failure is the transition from cata- strophic shear fracture to ductile necking of MG wires with reduction in wire diameter as reported in our previ- ous work17. Nevertheless, the 7% tensile elongation mediated by dense shear banding was not expected because zero tensile ductility of microscale MGs had been well founded in literatures1,31. The dense shear bands in Fig. 3e may be caused by retarding effect on shear band dynamics as in compressed bulk MG with Cu coating20–22. Transition from viscous fingering to dimpling. Results C ti In addition to the transition described above, transition from viscous fingering to dimpling was observed on fracture surfaces of the wires with different R values as shown in Fig. 4. As on fracture surfaces of various as-cast MG wires25,32–34, viscous fingering was found on the fracture surface of as-cast Pd40Cu30Ni10P20 MG wires as shown in Fig. 4a. The viscous fingering is an indication of Mode II fracture35,36 and a continuous crack propagation manner37. On the fracture surface of the MG wire core of the Cu-coated wire with an R value of 45%, both viscous fingering and dimples were found as shown in Fig. 4b, that is, the viscous fingering is changed into dimples at an R value around 45%. Even though the wire fractures in ScIentIfIc RePorTs \| (2018) 8:5659 \| DOI:10.1038/s41598-018-23956-5 4 www.nature.com/scientificreports/ Figure 5. Electrodepositing Cu onto Pd40Cu30Ni10P20 MG wire for tensile testing. (a) Schematic illustration of electrodeposition setup. The difference between our setup and conventional one for Cu electrodeposition is that a motor is connected to the cathode to rotate the MG wire to make the coating thickness homogeneous. (b) Cu coated Pd40Cu30Ni10P20 MG wire for tensile testing. The fillet between the grip section and the reduced section of the prepared tensile testing sample forms during electrodeposition because of the meniscus electrolyte around the MG wire at the position where the wire meets the surface of the electrolyte. Figure 5. Electrodepositing Cu onto Pd40Cu30Ni10P20 MG wire for tensile testing. (a) Schematic illustration of electrodeposition setup. The difference between our setup and conventional one for Cu electrodeposition is that a motor is connected to the cathode to rotate the MG wire to make the coating thickness homogeneous. (b) Cu coated Pd40Cu30Ni10P20 MG wire for tensile testing. The fillet between the grip section and the reduced section of the prepared tensile testing sample forms during electrodeposition because of the meniscus electrolyte around the MG wire at the position where the wire meets the surface of the electrolyte. catastrophic shear as shown in Fig. 3b, the MG wire core is confined by the Cu coating. Therefore, the final frac- ture of the wire is not in pure shear mode, and there may be a contribution of normal stress which causes the for- mation of dimples. With increasing R value, the fracture surfaces become rougher as shown in Fig. Discussion In order to investigate the effect of Cu coating on the tensile behavior of Cu-coated Pd40Cu30Ni10P20 MG wires, the tensile behavior of the coating was tested as shown in Fig. 2. The elastic limit is much lower than that of the as-cast MG wire, and the stress increases slowly with strain. Furthermore, Poisson’s ratio ν of metals undergo plastic deformation is 0.538. Therefore, we just simply take the Poisson’s ratio ν of the Cu coating as 0.5 to simplify theo- retical analysis. In addition, ν of Pd40Cu30Ni10P20 MG is 0.39939. The inner diameter of a tube or the diameter of a rod νε = − d d (1 ) 0 , where d0 is the original diameter, ε is the tensile strain. Hence, the inner diameter of a Cu tube is smaller than the diameter of a Pd40Cu30Ni10P20 MG rod with the same diameter d0. Therefore, there is pressure which is perpendicular to the loading direction and increases with increasing R. The pressure causes yielding strain of the MG core to increase with increasing R as shown in Fig. 3, because dependence of yielding strength of MG on pressure is negligible40,41. However, a literature42 has reported that plasticity of BMGs can be improved by pressure. This also applies to our experimental results that ductility increases with increasing pres- sure which increases with increasing R, as shown in Fig. 2. Because of the pressure, we believe that the dislocations in Cu coating sink at the Cu-MG interface, and a high volume fraction of crystalline phase means a high number of dislocation therefore a higher propensity for glassy phase to yield.hf g p p y g y p y The tensile behavior of Cu-coated Pd40Cu30Ni10P20 MG wire with different volume fraction of Cu coating R has been investigated. A maximum tensile elongation of 7.1% of the MG core has been achieved. SEM investigation has revealed that the tensile ductility is induced by multiple shear banding. Fracture of the wires transit from cata- strophic shear fracture to ductile fracture at an R value around 45%. At the same time, the pattern on the fracture surface of the MG core transits from viscous fingering to dimple. The pressure imposed on the surface of the MG core, which is induced by the Poisson’s ratio difference between the MG core and the Cu coating, is the reason of the ductility and the transitions. Results C ti 4c and d, and the stress states on the fracture surfaces of the MG wire cores become more complex. The rough surface would be caused by the intersecting shear bands showing in Fig. 3e, because the wires can no longer fracture along a single shear band. At the same time, the average dimple size of the wires with R values of 90% and 97% becomes smaller. Because of the rough fracture surfaces and complex stress states, the facture patterns shown in Fig. 4c and d cannot be analyzed theoretically by using physical models. Preparation of Cu-coated MG wires. The details of the preparation process of Pd40Cu30Ni10P20 MG wire can be found in our previous work24. The diameters of the prepared Pd40Cu30Ni10P20 MG wires range from 50 ∼ 100 μm. Discussion The pressure generated during deformation may be an effective way to tailor strength and ductility of materials. Methods Methods Preparation of Cu-coated MG wires. The details of the preparation process of Pd40Cu30Ni10P20 MG wire can be found in our previous work24. The diameters of the prepared Pd40Cu30Ni10P20 MG wires range from 50 ∼ 100 μm. ScIentIfIc RePorTs \| (2018) 8:5659 \| DOI:10.1038/s41598-018-23956-5 5 www.nature.com/scientificreports/ The electrolyte for Cu electrodeposition was prepared from copper sulfate pentahydrate (AR), sulfuric acid with a purity of 98 wt.%, and distilled water with 64 g/L Cu ion and 73.5 g/L sulfuric acid. The setup for Cu elec- trodeposition onto Pd40Cu30Ni10P20 MG wires is schematically shown in Fig. 5(a). A DC power supply was used to provide constant current. The anode was a copper-phosphorous (phosphorous content about 0.1 wt.%) plate with dimensions of 2 mm × 10 mm × 85 mm, and the cathode was a Pd40Cu30Ni10P20 MG wire. In order to ensure constant coating thickness, the cathode wire was clamped to the shaft of the motor as shown. The cathode was spinning at a speed of about 100 rpm during electrodeposition. The distance between the anode and cathode was kept at 30 mm. A magnetic stirrer with a spinning speed of about 120 rpm was used to stir the electrolyte. The current density i used in this work was 1 mA/mm2. The constant current provided by the power supply is I = iπd0l, where d0 is the diameter of the as-cast Pd40Cu30Ni10P20 MG wires, l is the length of the wire immersed in the elec- trolyte as shown in Fig. 5(a). In this work, the value of l was kept at a constant value of 70 mm. y g p In our experiments, the volume fraction of the Cu coating, given by = − R d d 1 / 0 2 1 2 (where d1 is the diameter of the Cu coated wires), needs to be controlled to specific values. The R was controlled by setting the electrodep- osition time t on the power supply. The volume of electrodeposited metal is proportional to I·t43, therefore, we used a commercial Ni wire with a diameter of 0.175 mm to measure the volume of Cu electrodeposited per sec- ond. The current I0 was calculated to be 0.038 A by using the equation above. After one-hour electrodeposition, the thickness of the wire became 0.321 mm. Then, the electrodeposition rate C0 was calculated to be 1.4 × 10−3 mm3/s. References References 1. Leamy, H. J., Wang, T. T. & Chen, H. S. Plastic flow and fracture of metallic glass. Metall. Trans. 3, 699–708 (1972). l 2. Conner, R. D., Johnson, W. L., Paton, N. E. & Nix, W. D. Shear bands and cracking of metallic glass plates in bending. J. Appl. Phys 94, 904–911 (2003). 94, 904–911 (2003). 3. Mukai, T., Nieh, T. G., Kawamura, Y., Inoue, A. & Higashi, K. Dynamic response of a Pd40Ni40P20 bulk metallic glass in tension. Scripta Mater. 46, 43–47 (2002). 3. Mukai, T., Nieh, T. G., Kawamura, Y., Inoue, A. & Higashi, K. Dynamic response of a Pd40Ni40P20 bulk metallic glass in tension Scripta Mater. 46, 43–47 (2002). p , ( ) 4. Wu, Y., Xiao, Y., Chen, G., Liu, C. T. & Lu, Z. Bulk metallic glass composites with transformation-mediated work-hardening and ductility. Adv. Mater. 22, 2770–2773 (2010). y ( ) 5. Hofmann, D. C. et al. Designing metallic glass matrix composites with high toughness and tensile ductility. Nature 451, 1085–1089 (2008). ( ) 6. Sha, Z. D., Pei, Q. X., Liu, Z. S., Zhang, Y. W. & Wang, T. J. Necking and notch strengthening in metallic glass with symmetric sharp- and-deep notches. Sci. Rep. 5, 10797 (2015). p p 7. Wang, Y., Li, J., Hamza, A. V. & Barbee, T. W. Ductile crystalline–amorphous nanolaminates. Proc. Natl. Acad. Sci. 104, 11155–11160 (2007).i 8. Lu, X. L., Lu, Q. H., Li, Y. & Lu, L. Gradient confinement induced uniform tensile ductility in metallic glass. Sci. Rep. 3, 3319 (2013) 9. Wang, Q. et al. Superior tensile ductility in bulk metallic glass with gradient amorphous structure. Sci. Rep. 4, 4757 (2014). 10. Qu, R. T., Zhang, Q. S. & Zhang, Z. F. Achieving macroscopic tensile plasticity of monolithic bulk metallic glass by surface treatment. Scripta Mater. 68, 845–848 (2013). p 11. Wang, H. et al. Relating residual stress and microstructure to mechanical and giant magneto- impedance properties in cold-drawn Co-based amorphous microwires. Acta Mater. 60, 5425–5436 (2012). p ( ) 2. Gao, M., Dong, J., Huan, Y., Wang, Y. T. & Wang, W. H. Macroscopic tensile plasticity by scalarizating stress distribution in bulk metallic glass. Sci. Rep. 6, 21929 (2016). g p 13. Sha, Z. D. et al. Metallic glass-based chiral nanolattice: Light weight, auxeticity, and superior mechanical properties. Mater. Today 20, 569–576 (2017). 14. Guo, H. et al. a availability. The data in this work are available from the corresponding authors on reasonable request. Data availability. The data in this work are available from the corresponding authors on reasonable request. Data availability. The data in this work are available from the corresponding authors on reasonable request. ScIentIfIc RePorTs \| (2018) 8:5659 \| DOI:10.1038/s41598-018-23956-5 Methods Therefore, the time needed to achieve a specific R value is t = Rπd0 2lI0/4I(1 − R).t h pi After electrodeposition of Cu with expected R, two end parts with lengths of 25 mm were electrodeposited with extra Cu to produce tensile testing sample shown in Fig. 5(b). When the surfaces of the wire meet the surface of the electrolyte, a meniscus of electrolyte forms around the wire. This is how the fillet forms between the grip section and the reduced section of the tensile sample, as shown in Fig. 5(b). Because of the fillet, the tensile testing sample fractured near the middle of the reduced section. In order to test tensile properties of the Cu coating, Cu layers with thickness of 0.6 mm were electroplated onto pure Al plates with dimensions of 2 mm × 10 mm × 70 mm with the same electroplating parameters described above. After electroplating, the Al plates were removed by using a NaOH solution, and the Cu layers were machined into dog-bone tensile testing specimen, with gauge dimensions of 0.6 mm × 3 mm × 15 mm. Electropolishing of Cu-coating. In order to investigate the deformation morphology of the MG core of the Cu-coated MG wires, the Cu-coating was electropolished before SEM observation. The setup in Fig. 5a was used for electropolishing. However, the Cu-coated sample was set as the anode and the Cu plate was set as the cathode. The electrolyte used was the same as the electrolyte for electrodeposition. The motor did not spin during the electrodeposition. The power supply was working in the constant voltage mode, with a voltage of 0.5 V to electropolish the Cu Coatings. 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J.Y. and K.C.C. initiated the project, I.H. and Y.Y.J. performed the experiments, J.Y., I.H., K.C.C., Y.Y.J., Y.D.J., G.W., and Q.J.Z. contributed to data analyzing, manuscript writing and discussion. Acknowledgementsh g This work was supported by the Research Committee of the Polytechnic University of Hong Kong (Project code: ) l l f h ( d ) d h l This work was supported by the Research Committee of the Polytechnic University of Hong Kong (Project code: G-YBE1) National Natural Science of China (Grant Nos 51501106 and 51771106) and the Natural Science This work was supported by the Research Committee of the Polytechnic University of Hong Kong (Project code: G-YBE1), National Natural Science of China (Grant Nos 51501106 and 51771106), and the Natural Science Foundation of Shanghai (Grant No. 17ZR1440800). The authors would like to thank Dr. Shunhua Chen and Prof. Qing Wang for experimental help and discussion. 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Cooling rate-dependent yield behavior of metallic glass wires. Mater. Sci. Eng. A 683, 236–243 (2017). 26. Lu, L., Shen, Y., Chen, X., Qian, L. & Lu, K. Ultrahigh strength and high electrical conductivity in copper. Science 304, 422 (2004). ( ) 27. Thomas H, C. ScIentIfIc RePorTs \| (2018) 8:5659 \| DOI:10.1038/s41598-018-23956-5 Additional Informationh Competing Interests: The authors declare no competing interests. Competing Interests: The authors declare no competing interests. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps an institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre- ative Commons license, and indicate if changes were made. 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https://openalex.org/W3122595189	https://www.researchsquare.com/article/rs-146523/latest.pdf	English	null	Mathematical Model of Human Semicircular Canal Spatial Attitude&nbsp;	Research Square (Research Square)	2,021	cc-by	6,138	Mathematical Model of Human Semicircular Canal Spatial Attitude Mathematical Model of Human Semicircular Canal Spatial Attitude Shu-zhi Wu Third A¨liated Hospital of Shanghai University Ping Lin Third A¨liated Hospital of Shanghai University Yan-yan Zheng Third A¨liated Hospital of Shanghai University Yi-fei Zhou Third A¨liated Hospital of Shanghai University Xiao-kai Yang (  yakeworld@126.com ) Third A¨liated Hospital of Shanghai University Research Article Posted Date: January 19th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-146523/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Mathematical model of human semicircular canal spatial attitude Wu Shu-zhi, Lin Ping, Zheng Yan-yan, Zhou Yi-fei, Yang Xiao-kai* Wu Shu-zhi, the Wenzhou Third Clinical Institute Affiliated to Wenzhou Medical University/the Third Affiliated Hospital of Shanghai University/the Wenzhou People’s Hospital, Neurology Dept, Wenzhou, Zhejiang, China Lin Ping, the Wenzhou Third Clinical Institute Affiliated to Wenzhou Medical University/the Third Affiliated Hospital of Shanghai University/the Wenzhou People’s Hospital, Neurology Dept, Wenzhou, Zhejiang, China Zheng Yan-yan, the Wenzhou Third Clinical Institute Affiliated to Wenzhou Medical University/the Third Affiliated Hospital of Shanghai University/the Wenzhou People’s Hospital, Neurology Dept, Wenzhou, Zhejiang, China Zhou Yi-fei, the Wenzhou Third Clinical Institute Affiliated to Wenzhou Medical University/the Third Affiliated Hospital of Shanghai University/the Wenzhou People’s Hospital, ENT Dept, Wenzhou, Zhejiang, China Yang Xiao-kai, the Wenzhou Third Clinical Institute Affiliated to Wenzhou Medical University/the Third Affiliated Hospital of Shanghai University/the Wenzhou People’s Hospital, Neurology Dept, Wenzhou, Zhejiang, China Wu Shu-zhi, Lin Ping, Zheng Yan-yan, Zhou Yi-fei, Yang Xiao-kai* Wu Shu-zhi, Lin Ping, Zheng Yan-yan, Zhou Yi-fei, Yang Xiao-kai* Wu Shu-zhi, the Wenzhou Third Clinical Institute Affiliated to Wenzhou Medical University/the Third Affiliated Hospital of Shanghai University/the Wenzhou People’s Hospital, Neurology Dept, Wenzhou, Zhejiang, China Lin Ping, the Wenzhou Third Clinical Institute Affiliated to Wenzhou Medical University/the Third Affiliated Hospital of Shanghai University/the Wenzhou People’s Hospital, Neurology Dept, Wenzhou, Zhejiang, China Zheng Yan-yan, the Wenzhou Third Clinical Institute Affiliated to Wenzhou Medical University/the Third Affiliated Hospital of Shanghai University/the Wenzhou People’s Hospital, Neurology Dept, Wenzhou, Zhejiang, China Zhou Yi-fei, the Wenzhou Third Clinical Institute Affiliated to Wenzhou Medical University/the Third Affiliated Hospital of Shanghai University/the Wenzhou People’s Hospital, ENT Dept, Wenzhou, Zhejiang, China Yang Xiao-kai, the Wenzhou Third Clinical Institute Affiliated to Wenzhou Medical University/the Third Affiliated Hospital of Shanghai University/the Wenzhou People’s Hospital, Neurology Dept, Wenzhou, Zhejiang, China Wu Shu-zhi, the Wenzhou Third Clinical Institute Affiliated to Wenzhou Medical University/the Third Affiliated Hospital of Shanghai University/the Wenzhou People’s Hospital, Neurology Dept, Wenzhou, Zhejiang, China Lin Ping, the Wenzhou Third Clinical Institute Affiliated to Wenzhou Medical University/the Third Affiliated Hospital of Shanghai University/the Wenzhou People’s Hospital, Neurology Dept, Wenzhou, Zhejiang, China Yang Xiao-kai, the Wenzhou Third Clinical Institute Affiliated to Wenzhou Medical University/the Third Affiliated Hospital of Shanghai University/the Wenzhou People’s Hospital, Neurology Dept, Wenzhou, Zhejiang, China Corresponding Author: Yang Xiao-kai No. 57 Canghou Street, Wenzhou, Zhejian, China 325000 Phone: 0086-0577-88053656 yakeworld@126.com https://orcid.org/0000-0002-8189-2017 Corresponding Author: Yang Xiao-kai No. 57 Canghou Street, Wenzhou, Zhejian, China 325000 Phone: 0086-0577-88053656 yakeworld@126.com https://orcid.org/0000-0002-8189-2017 Wu Shu-zhi 45587732@qq.com https://orcid.org/0000-0002-4907-7127 Lin Ping 33106846@qq.com https://orcid.org/0000-0002-8361-378X Zheng Yan-yan 86504540@qq.com https://orcid.org/0000-0002-7797-7229 Zhou Yi-fei yifa31wz@163.com https://orcid.org/0000-0002-2065-1420 Financial Disclosure : Wu Shu-zhi - Reports no disclosures Lin Ping - Reports no disclosures Zheng Yan-yan - Reports no disclosures Zhou Yi-fei - Reports no disclosures Yang Xiao-kai - Reports no disclosures Study Funded by Wenzhou Municipal Science and Technology Bureau [Grant No. ZS2017020,Y2020420 ], Joint Funds of the Zhejiang Province Natural Science Foundation of China [Grant No. LSY19H090002], and Foundation of Zhejiang Provincial Science and Technology Bureau [Grant No. LGF18H090007]. 1 ABSTRACT Located deep in the temporal bone, the semicircular canal is a subtle structure that requires a spatial coordinate system for measurement and observation. In this study,Fifty-five semicircular canal and eyeball models were obtained by segmentation of MRI data. The spatial coordinate system was established by taking the top of the common crus and the bottom of eyeball as the horizontal plane. Firstly, the plane equation is calculated according to the centerline of the semicircular canals. Then, according to the parameters of the plane equation, the plane normal vectors are obtained. Finally, the average unit normal vector of each semicircular canal plane can be obtained by calculating the average value of the vectors. It is more intuitive and accurate to calculate the average normal vector of semicircular canal plane with the vector average method, which is different from the angular average method in different degrees. The mathematical model of semicircular canal spatial attitude established in this study is more reliable, which can guide the vestibular function examination, and also help guide the diagnosis and treatment of BPPV. [key words] Semicircular canal; Measurement; Spatial direction; Magnetic resonance imaging; Human; Model Introduction The position and direction of semicircular canals in three-dimensional coordinate systems are very important for vestibular function examination and BPPV diagnosis and treatment. Because the semicircular canal is located in the deep part of the temporal bone, its structure is complicated and delicate, which makes it difficult to observe and measure directly. Moreover, it is necessary to establish a spatial coordinate system to measure the spatial direction of the semicircular canal, which makes it rare to study the spatial attitude of the semicircular canal. In addition, the reported data are often inconsistent1,2,3,4, often lack of reliable spatial coordinate system, and are limited to the study of semicircular canal morphology and the relative position relationship between the semicircular canal5,6,7,8,9,10,11. With the development of modern medical imaging and computer technology, the planar fitting and spatial posture measurement of semicircular canals based on 3D reconstruction technology have become the research focus of semicircular canal anatomy and morphology. Bradshaw studies the spatial posture of the semicircular canal through semi-automatic segmentation of semicircular canal, automatic extraction of semicircular canal centerline, and calculation of semicircular canal plane fitting equation. This method is more reliable than manual measurement reported in other literature, but its biggest disadvantage is that the head space coordinate system is not established. Because the plane equations of each semicircular canal are not in the standard spatial coordinate system, the application of the measurement results is limited7. The author explains that the reason why the head space coordinate system was not established is that it is difficult to determine the landmark positions needed for establishing the coordinate system, and the scanning range is insufficient, resulting in the omission of the landmark position. The commonly used three-dimensional coordinate systems include Frankfurt coordinate system4,11 and Reid's coordinate system3. The commonly used three-dimensional coordinate systems include Frankfurt coordinate system4,11 and Reid's coordinate system3. y kfort horizontal is the plane passing through bilateral porions and the left orbitale. 2 Reid’s plane is defined as the plane that passes through the center of each external auditory canal and the inferior margin of the two orbits1,12. Reid’s plane is defined as the plane that passes through the cen auditory canal and the inferior margin of the two orbits1,12. It is more difficult to find bone markers on MRI images. segmentation for semicircular canal and eyeball The boundary between the eyeball and the surrounding tissues is clear, and the method based on threshold can quickly segment the eyeball. However, because the thresholds of the semicircular canal and cochlea are different, it is difficult to segment the inner ear based on the threshold method. The Segment Editor module of 3D slicer includes threshold segmentation function and provides a variety of automatic thresholds searching methods. Automatic threshold searching methods, such as the Otsu method, can segment the semicircular canal, but the model surface is not smooth enough. To improve the segmentation result, the markers of segmentation results are expanded and extracted into the voxel model, and then transformed into the surface model by using the marching cubes function of the grayscale model maker, which makes the surface of the semicircular canal model smoother. Understanding of semicircular canal morphology 1. Identification of semicircular canal Introduction Some studies have tried to use semicircular canals and eyeballs to establish a spatial coordinate system, and it is considered that the plane formed by the bifurcation of the common crus and the center of the eyeball is horizontal13,14. y However, further studies have shown that the plane formed by the bifurcation of the common crus and the bottom of the eyeball is parallel to the Frankfurt plane15. In previous studies, the angle between the plane of the semicircular canal and the coordinate planes were calculated first, and then the average value of each angle was calculated. y However, further studies have shown that the plane formed by the bifurcation of the common crus and the bottom of the eyeball is parallel to the Frankfurt plane15. y p p In previous studies, the angle between the plane of the semicircular canal and the coordinate planes were calculated first, and then the average value of each angle was calculated. As a matter of fact, the spatial direction of the semicircular canal is determined by the normal vector of the semicircular canal. The coordinate value of the unit vector of the semicircular canal plane normal vector is equal to the cosine value of the direction angle. According to the rules of vector calculation, the average value of normal vectors in the semicircular canal plane should be the average value of the cosine of direction angle, not the average value of the direction angle. In this study, firstly, the spatial coordinate system is established through the semicircular canal and eyeball, then the plane equation is fitted according to the centerline of the semicircular canal and the normal vector is obtained. Finally, the mathematical model of the spatial posture of the human semicircular canal based on the average unit normal vector of each semicircular canal plane is established, which is convenient for vestibular research and application. 1. Identification of semicircular canal The centerline of the inner ear can show its morphological characteristics. First, the intersection point and the line segments between the intersections points are analyzed. According to the length of the line segments, three semicircular canal central rings and a straight line from the cochlea to utricle can be extracted. The length of the centerline of the posterior semicircular canal, the superior semicircular canal and the lateral 3 3 semicircular canal decreased in turn, but sometimes the connection from the cochlea to the utricle and other abnormal connections affected the judgment. Specific anatomical structures can be distinguished according to the relative spatial position and length of the lines. The midpoint of the central ring of the posterior semicircular canal is in the rearmost position, the midpoint of the central ring of the superior semicircular canal is in the uppermost position, the midpoint of the central ring of the the lateral semicircular canal is in the outermost position, and the midpoint of the connecting line from the cochlea to the utricle is in the foremost position. 2. Identify critical intersections: The semicircular canal model obtained by semi-automatic segmentation is consistent in shape. Most of the central line of the semicircular canal include four key points, including point A( bifurcation of the common crus) which connecting posterior semicircular canal (AD) and anterior semicircular canal (AC); point C which connecting superior semicircular canal (CA), lateral semicircular canal (CB) and utricle (E), B and D may coincide; point D which connects posterior semicircular canal (DA) and elliptic sac (DE). (Fig.1) Figure 1 ( a ) ( b ) Figure 1 Centerline and key points of semicircular canal (a) The centerline of the semicircular canal is composed of a set of points; (b) Most centerlines include four key points: A, B, C and D. A is the bifurcation of the common crus, AD is the posterior semicircular canal, AC is the anterior semicircular canal, BC is the lateral semicircular canal, and point E is located in the Utricle ( a ) ( b ) Centerline and key points of semicircular canal Centerline and key points of semicircular canal (a) The centerline of the semicircular canal is composed of a set of points; (b) Most centerlines include four key points: A, B, C and D. A is the bifurcation of the common crus, AD is the posterior semicircular canal, AC is the anterior semicircular canal, BC is the lateral semicircular canal, and point E is located in the Utricle 4 The superior intersection of the posterior semicircular canal and the superior semicircular canal is the top of the common corus (A), and then other key points D, C, B and E can be identified in turn. 3. Mathematical model of space attitude of semicircular canal The average unit normal vector of each semicircular canal plane is obtained by calculating the sum of the normal vectors of 55 pairs of posterior semicircular canal plane, superior semicircular canal plane, and lateral semicircular canal plane(Fig.2). Figure 2 semicircular canal plane fitting (a) Fitting spline curve of semicircular canal centerline (b) Fitting plane of semicircular canal according to spline curve. (a) (b) (a) (b) (a) (b) semicircular canal plane fitting semicircular canal plane fitting (a) Fitting spline curve of semicircular canal centerline (b) Fitting plane of semicircular canal according to spline curve. (a) Fitting spline curve of semicircular canal centerline (b) Fitting plane of semicircular canal according to spline curve. Because their spatial coordinates are the cosine values of the angles along X, Y, and Z axes of the standard spatial coordinate system, the angles between the semicircular canal plane and the corresponding sagittal plane, coronal plane, and horizontal plane can be calculated by using arccosine function (see Table 1). Table 1: calculate the angle between the plane of semicircular canal and the coordinate plane by Vector method 5 Semicircular canal Angle with sagittal plane(°) Angle with coronal plane(°) Angle with horizontal plane(°) Deviation range(x±s°) psp_r 130.62 45.37 73.29 5.26±2.52 asp_r 41.47 54.74 71.07 5.31±2.69 hsp_r 90.95 107.38 17.41 6.94±3.64 psp_l 48.67 45.39 74.57 5.40±2.92 asp_l 137.62 53.96 70.80 5.60±2.98 hsp_l 88.55 106.18 16.25 6.73±3.15 psp=posterior semicircular canal; asp= Superior semicircular canal; hsp= Lateral semicircular canal; r=right;l=left The unit normal vector of each semicircular plane is distributed on the circle with radius 1. For the convenience of observation, the radius is expanded to 100, the normal vectors of different semicircular canal planes are marked with different color points, and the average normal vectors are represented by arrows. The distribution and relationship of normal vectors of semicircular canal plane can be observed directly (Fig.3). Figure 3 Figure 3 Distribution and average normal vector of semicircular canal plane.The normal vectors of bilateral lateral semicircular canal planes are nearly parallel, and the normal vectors of unilateral posterior semicircular canal and contralateral superior semicircular canal planes are nearly parallel. The distribution area of normal vector of each semicircular plane is concentrated. (a) ovewhead view (b) back view PSC = posterior semicircular canal, ASC = superior semicircular canal, HSC = lateral semicircular canal, R = Right, L = Left ( b ) ( a ) ( b ) ( a ) Distribution and average normal vector of semicircular canal plane.The normal vectors of bilateral lateral semicircular canal planes are nearly parallel, and the normal vectors of unilateral posterior semicircular canal and contralateral superior semicircula canal planes are nearly parallel. The distribution area of normal vector of each semicircular plane is concentrated. semicircular canal plane fitting (a) ovewhead view (b) back view Distribution and average normal vector of semicircular canal plane.The normal vectors of bilateral lateral semicircular canal planes are nearly parallel, and the normal vectors of unilateral posterior semicircular canal and contralateral superior semicircular canal planes are nearly parallel. The distribution area of normal vector of each semicircular plane is concentrated. (a) ovewhead view (b) back view PSC = posterior semicircular canal, ASC = superior semicircular canal, HSC = lateral semicircular canal, R = Right, L = Left The traditional method is to calculate the angle between the semicircular canal plane and the coordinate plane first, and then calculate the average value of the angle (see Table 2) 6 Table 2: calculate the angle between the plane of semicircular canal and the coordinate plane by average angle method Semicircular canal Angle with sagittal plane(x±s°) Angle with coronal plane(x±s°) Angle with horizontal plane(x±s°) psp_r 130.50±4.12 45.50±4.36 73.34±3.97 asp_r 41.59±4.83 54.84±4.43 71.13±3.63 hsp_r 90.95±5.92 107.28±5.08 18.33±5.20 psp_l 48.82±4.21 45.57±4.00 74.61±4.61 asp_l 137.44±4.59 54.12±4.00 70.85±4.75 hsp_l 88.55±5.89 106.09±4.5 17.24±4.57 psp=posterior semicircular canal; asp= Superior semicircular canal; hsp= Lateral semicircular canal; r=right;l=left When the data distribution is more concentrated, the results of vector averaging method and angular averaging method are similar; when the data distribution is more dispersed, the difference between the results of vector averaging method and angular averaging method will increase. Discussions The organ function is always precisely correlated with its anatomical features. However, unlike other organs, the study of semicircular canal function needs not only to understand the anatomical and morphological characteristics of the semicircular canal but also to understand its position and direction in three-dimensional space. In recent years, with the development of vertigo science, especially the need for diagnosis and treatment of BPPV, the knowledge of semicircular canal space posture is urgently needed. In the past, the study of semicircular canal anatomy usually used cadavers, which was limited to anatomical morphology, and the spatial posture of the semicircular canal was not fully studied. In recent years, with the development of medical imaging, it is possible to obtain a semicircular canal model by segmenting temporal bone image data to study the anatomical morphology and spatial posture of the semicircular canal. The prerequisite for measuring the spatial orientation of the semicircular canal is to establish a spatial coordinate system. Commonly used three-dimensional spatial coordinate systems include the Frankfurt coordinate system 11 and Reid's coordinate system 3, which is determined by bony points including the superior or midpoint of the auricle and the infra-orbital margin point. The study of the spatial posture of the semicircular canal has been limited by the lack of scan coverage that did not include the bony points, or by the difficulty of identifying the bony points on MRI scans, making it difficult to establish a spatial coordinate system. It is found that the plane formed by common crus bifurcation and eyeball bottom is parallel to the Frankfurt plane, which makes it possible to establish a spatial coordinate system through the semicircular canal and eyeball, and is especially suitable for the automatic construction of standard spatial coordinate systems15,. To measure the spatial direction of semicircular canals, the plane of the semicircular 7 canal needs to be obtained. There are different methods to establish the semicircular canal plane, including taking three coordinates at different positions of the semicircular canal, taking the semicircular canal bisector plane, and taking the semicircular canal centerline. Among them, the most scientific method is to automatically take the centerline of the semicircular canal to construct the semicircular canal plane7,. Discussions Compared to the average value of direction angle (Table 2), the method of calculating the average value of the semicircular canal plane normal vector (Table 1) is more intuitive and accurate, and the results are not completely consistent, especially for the data of lateral semicircular canal. The possible reason is that the length of lateral semicircular canal is short and has a certain curvature, and the individual difference of plane fitting is large. This may be because the shortest and non-planar nature of the lateral semicircular canal leads to the increase of individual differences in plane fitting 6, 7, which can lead to inaccurate results from the mathematical average method of direction angular value. Moreover, the understanding of the spatial direction of the semicircular canal is often based on an intuitive understanding of the angle between two straight lines in a two-dimensional plane. For example, if the angle between the posterior semicircular canal and the sagittal plane is 45 degrees, it is considered that the posterior semicircular canal is parallel to the sagittal plane after rotating around the Z-axis for 45 degrees. This problem can also be transformed into the rotation of the normal vector of the semicircular canal plane. In the two-dimensional plane, the angle between the unit vector v1 → and the unit vector v2 → is θ and it is right that the vector v1 → is parallel to the vector v2 → after rotation angle θ . Viewed in three dimensions, the axis of rotation is the normal vector u⃗ of the plane formed by the vector v1 → and the vector v2 → . Similarly, the angle between the posterior semicircular canal and the sagittal plane is 45 degrees. To make the posterior semicircular canal rotate and parallel to the sagittal plane, that is, to make the posterior semicircular canal plane normal vector v1 → rotate and parallel to the sagittal plane normal vector v2 → (X-axis)，it should rotate 45 degrees around the vector u⃗ , which is the normal vector of the plane formed by vector v1 → and vector v2 → , instead of rotating around the Z-axis. According to the angle between the plane of semicircular canal and the coordinate plane in Table 1, the unit vector of the plane normal vector can be constructed, and the coordinate value is the cosine value of the direction angle. Discussions Skeletonize's skeletonize function supports 3D image skeleton extraction, but the 3D Slicer software has a VMTK plug-in, which can call its vtkvmtkPolyDataCenterline function in python programming to get the centerline of the inner ear model, and can observe and analyze it in the 3D Slicer software. Traditionally, different studies on measuring the spatial direction of the semicircular canal always calculate the angles between each semicircular canal and the sagittal plane, coronal plane, and cross-sectional plane, and then take the average value of the angles in each coordinate plane as the spatial direction of the semicircular canal(Table 2) Set the unit normal vector of the semicircular canal based on averaging angles to be nu ⃗⃗⃗⃗ then nu → = (cos ∑ n i=1 αi n ) i + (cos ∑ n i=1 βi n) j + (cos ∑ n i=1 γi n) k Where n is the number of semicircular canal planes, αi, βi, γi is the direction angle of the ith semicircular canal, and I, j, k are coordinate unit vectors. The correctness of this method needs further discussion and verification. According to the dihedral angle principle, the angle between the two planes is equal to the angle between the normal vectors or the complementary angle of the included angle. In this way, the spatial direction measurement of the semicircular canal can be transformed into the problem of its normal vector direction angle. The unit normal vector of the semicircular canal plane is a three-dimensional space vector, and the coordinate value of the unit vector is the cosine value of its direction angle. Unlike the calculation of the mean direction angle, the mean value of the vector are calculated as the mean value of the cosine of the directional angle (see Table 1), i.e., to sum the unit normal vectors of each semi-regulated plane to find it's unit normal vector nu → , that is α = ∑ n 𝑖=1 cos αi β = ∑ n 𝑖=1 cos βi 8 8 γ = ∑ n i=1 cos γi Z = √α2 + β2 + γ2 nu → = α Z i + β Z j + γ Z k Where n is the number of semicircular canal planes, α_i, β_i, γ_i is the directional angle of the ith semicircular canal, i, j, k is the coordinate unit vector. Discussions If you need to rotate the posterior semicircular canal to make it parallel to the sagittal plane, you only need to rotate its plane normal vector (blue line) to make it parallel to the X- axis, that is, the sagittal normal vector(Fig.4). 9 Fig. 4 Solid model of human semicircular canal spatial attitude Red, blue, and green represent the normal vectors of the right lateral semicircular canal plane, posterior semicircular canal plane and superior semicircular canal plane, respectively. The medial cross represents the Y-axis and Z-axis. Fig. 4 Solid model of human semicircular canal spatial attitude Fig. 4 Solid model of human semicircular canal spatial attitude Red, blue, and green represent the normal vectors of the right lateral semicircular canal plane, posterior semicircular canal plane and superior semicircular canal plane, respectively. The medial cross represents the Y-axis and Z-axis. Fig. 4 Solid model of human semicircular canal spatial attitude Red, blue, and green represent the normal vectors of the right lateral semicircular canal plane, posterior semicircular canal plane and superior semicircular canal plane, respectively. The medial cross represents the Y-axis and Z-axis. Most of the previous studies are based on manual point taking and measurements, which cannot meet the need to know the spatial attitude of semicircular canals accurately. Therefore, we use automatic semicircular plane fitting technology, use means of vector instead of means of direction angle to obtain semicircular spatial orientation, and establish a mathematical model of semicircular spatial attitude, which can guide the vestibular function examination such as head shaking test, and also help to guide the diagnosis and treatment of BPPV. Examination methods Using Siemens 1.5T superconducting magnetic resonance system and standard head coil for inner ear examination. 3D constructive interference insteady state sequence(3D- CISS) (TR: 6.0ms, TE: 2.7ms, FOV:135mmX180mm, Matrix: 256X192, Thickness: 0.7mm) was performed. Clinical data Clinical data 55 cases included in the study with normal inner ear examined by MRI during the period from January 2014 to December 2019. Inclusion criteria: (1) the semicircular canal was clearly displayed without artifacts; (2) the bottom of the eyeball was clearly without artifacts; (3) a complete semicircular canal model could be obtained by image segmentation. Exclusion criteria: (1) the existence of local lesions may affect the anatomical structure of semicircular canals; (2) the presence of abnormal head structure. Image processing and modeling The original image data were exported from PACS and saved in DICOM format. The images were obtained by reading the catalog with 3D slicer 4.10.2 software, and the 3D- CISS Sequence was automatically exported and saved in NII format. 55 cases of semicircular canal and eyeball were semi-automatically segmented and exported to STL format。 Methods 10 Analysis of space attitude of semicircular canal 1.Obtain the centerline of semicircular canal VMTK is an open-source c + + library using ITK and VTK for vascular structure segmentation, extraction, and analysis. It has a plug-in for 3D Slicer software. It mainly uses its vtkvmtkPolyDataCenterline function to get the inner ear centerline model16. The centerline model of the inner ear is converted to a series of points.It can be seen from the analysis that the centerline of the whole inner ear includes three semicircular canal central rings. There are common intersections between the rings. The intersections appear at least three times in the array. The distance between the intersections of the semicircular canal central rings has a fixed pattern, according to which other abnormal intersections can be excluded. The centerline of semicircular canal can show the morphological characteristics of semicircular canal. According to the relative spatial position of the central ring of the semicircular canal, we can identify each semicircular canal, that is, the posterior semicircular canal is in the rearmost position, the superior semicircular canal is in the uppermost position, and the lateral semicircular canal is in the outermost position. The superior intersection of the posterior semicircular canal and the superior semicircular canal is the top of the common crus. 11 2.Establishment of the standard spatial coordinate system 2.Establishment of the standard spatial coordinate system Firstly, the fundus plane was formed by taking the lowest point of the eyeball (the minimum Z value) and the bifurcation of bilateral common crus, and the mathematical equation of the plane was calculated. Firstly, the fundus plane was formed by taking the lowest point of the eyeball (the minimum Z value) and the bifurcation of bilateral common crus, and the mathematic equation of the plane was calculated. Then, adjusting the plane is needed. The distance from each point of the eyeball to the plane can be calculated by the mathematical equation of the plane with the coordinate values of the point. The point with the longest distance is taken as the lowest point of the eyeball in the standard coordinate system, which forms a horizontal plane with the bifurcation of bilateral common crus. To establish the spatial coordinate system, the coordinate axes must be determined. Ethical approval This study was conducted in accordance with the Helsinki protocol and standard of Good Clinical Practice, and was approved by the Ethics Committee of the Wenzhou People’s Hospital (No.2018143). The Ethics Committee determined that the study was exempt from an informed consent requirement since it was a review of existing clinical data with patient identifiers removed. References [1] Blanks, R. H. I., Curthoys, I. S. & Markham, C. H. Planar relationships of the semicircular canals in man. Acta Otolaryngol. (Stockh.). 1-6, 185-196 (1975). [2] Della Santina, C. C., Potyagaylo, V., Migliaccio, A. A., Minor, L. B. & Carey, J. P. Orientation of human semicircular canals measured by three-dimensional multiplanar CT reconstruction. J. Assoc. Res. Otolaryngol.. 3, 191-206 (2005). [3] Hashimoto, S., Naganuma, H., Tokumasu, K., Itoh, A. & Okamoto, M. Three-dimensional reconstruction of the human semicircular canals and measurement of each membranous canal plane defined by Reid's stereotactic coordinates. Annals of Otology, Rhinology & Laryngology. 12, 934-938 (2005). [4] Lyu, H.-Y. et al. The age-related orientational changes of human semicircular canals. Clinical an Experimental Otorhinolaryngology. 2, 109 (2016). [5] Cox, P. G. & Jeffery, N. Geometry of the semicircular canals and extraocular muscles in rodent lagomorphs, felids and modern humans. J. Anat.. 5, 583-596 (2008). [6] Sato, H., Sando, I., Takahashi, H. & Fujita, S. Torsion of the human semicircular canals and its influence on their angular relationships. Acta Otolaryngol. (Stockh.). 2, 171-175 (1993). [7] Bradshaw, A. P. et al. A mathematical model of human semicircular canal geometry: a new basis for interpreting vestibular physiology. J. Assoc. Res. Otolaryngol.. 2, 145-159 (2010). [8] Lee, J. et al. A morphometric study of the semicircular canals using micro‐CT images in three‐ dimensional reconstruction. The Anatomical Record. 5, 834-839 (2013). [9] El Khoury, M. et al. The human semicircular canals orientation is more similar to the bonobos than to the chimpanzees. PLoS One. 4, e93824 (2014). [10] Kim, D. K. et al. Analysis of the coplanarity of functional pairs of semicircular canals using three- dimensional images reconstructed from temporal bone magnetic resonance imaging. The Journal of Laryngology and Otology. 5, 430 (2015). [11] Yılmazer, R. & Topçuoğlu, O. M. Three-Dimensional Reconstruction of the Semicircular Canals with a Two-Hands Model. Turkish Archives of Otorhinolaryngology. 4, 176 (2019). [12] Kimura, M. et al. Reliability of a coordinate system based on anatomical landmarks of the maxillofacial skeleton: an evaluation method for three-dimensional images obtained by cone- beam computed tomography. Oral Radiology. 1, 37-42 (2009). [13] Suzuki, K., Masukawa, A., Aoki, S., Arai, Y. & Ueno, E. A new coordinates system for cranial organs using magnetic resonance imaging. Acta Otolaryngol. 5, 568-575 (2010). [14] Aoki, S., Takei, Y., Suzuki, K., Masukawa, A. & Arai, Y. Data files are available on request to the corresponding author. Data files are available on request to the corresponding author. Analysis of space attitude of semicircular canal The normal vector of the horizontal plane is Z axis Because the semicircular canals are symmetrical left and right, the line connecting the bilateral crus bifurcation is selected as the X-axis, and the Y-axis is further determined as the cross product of the X-axis and Z-axis. In order to keep consistent with the spatial coordinate system of 3D slicer software, the right-hand Cartesian coordinate system is adopted. 3.Fitting equation of semicircular canal plane The centerline of the semicircular canal can be transformed into a group of points with different adjacent distances. The distribution of the points in the curved part is dense, while that in the straight part is sparse. It is inaccurate to fit the plane directly according to these points. In order to improve the fitting results, firstly, the spline curves were fitted according to the central line of the semicircular canal, and then the points of the spline curves were calculated according to the least square method to fit the semicircular canal plane. To determine its position in the standard spatial coordinate system, it is necessary to further calculate the angle(direction angle) between its normal vector and each axis in the standard spatial coordinate system and then establish the unit normal vector in the standard space coordinate system according to the cosine of the direction angle (direction cosine). Presume the plane normal vector of semicircular canal as a = {x, y, z } , and the angles between it and the x, y, z axes of the standard space coordinate system are respectively α, β, γ,and the vector a° is the unit normal vector of the vectorv a in the standard space coordinate system, Then a° = (cosα)i + (cosβ)j + (cosγ)k, where i,j,k are unit vector of coordinate system. 4.Establish the mathematical model of semicircular canal attitude Firstly, the plane equations of the posterior semicircular canal, the superior semicircular canal, and the lateral semicircular canal are calculated, and the unit vector of the normal vector in the standard spatial coordinate system is obtained. Finally, the average unit normal vector of each semicircular canal plane needs to be calculated to form the mathematical model of the spatial posture of the human semicircular canal. 12 References Planer orientation of the bilater semicircular canals in dizzy patients. Auris. Nasus. Larynx. 5, 451-454 (2012). [15] Chen, X. S., Lin, Y. J., Yang, X. G., Lin, D. & Yang, X. K. A comparative analysis of the semicircula canal and eyeball plane and the Frankfort horizontal plane. Zhejiang Clinical Medical Journal. 20, 821-823 (2018). [16] Izzo, R., Steinman, D., Manini, S. & Antiga, L. The vascular modeling toolkit: a Python library for the analysis of tubular structures in medical images. Journal of Open Source Software. 25, 745 (2018). 13 Figures Figures g Figure 1 Centerline and key points of semicircular canal (a) The centerline of the semicircular canal is composed of a set of points; (b) Most centerlines include four key points: A, B, C and D. A is the bifurcation of the common crus, AD is the posterior semicircular canal, AC is the anterior semicircular canal, BC is the lateral semicircular canal, and point E is located in the Utricle Figure 1 Centerline and key points of semicircular canal (a) The centerline of the semicircular canal is composed of a set of points; (b) Most centerlines include four key points: A, B, C and D. A is the bifurcation of the common crus, AD is the posterior semicircular canal, AC is the anterior semicircular canal, BC is the lateral semicircular canal, and point E is located in the Utricle Centerline and key points of semicircular canal (a) The centerline of the semicircular canal is composed of a set of points; (b) Most centerlines include four key points: A, B, C and D. A is the bifurcation of the common crus, AD is the posterior semicircular canal, AC is the anterior semicircular canal, BC is the lateral semicircular canal, and point E is located in the Utricle Figure 2 Figure 2 Figure 2 semicircular canal plane ¦tting (a) Fitting spline curve of semicircular canal centerline (b) Fitting plane of semicircular canal according to spline curve. semicircular canal plane ¦tting (a) Fitting spline curve of semicircular canal centerline (b) Fitting plane of semicircular canal according to spline curve. Figure 3 Distribution and average normal vector of semicircular canal plane.The normal vectors of bilateral lateral semicircular canal planes are nearly parallel, and the normal vectors of unilateral posterior semicircular canal and contralateral superior semicircular canal planes are nearly parallel. The distribution area of normal vector of each semicircular plane is concentrated. (a) ovewhead view (b) back view PSC = posterior semicircular canal, ASC = superior semicircular canal, HSC = lateral semicircular canal, R = Right, L = Left Distribution and average normal vector of semicircular canal plane.The normal vectors of bilateral lateral semicircular canal planes are nearly parallel, and the normal vectors of unilateral posterior semicircular canal and contralateral superior semicircular canal planes are nearly parallel. The distribution area of normal vector of each semicircular plane is concentrated. (a) ovewhead view (b) back view PSC = posterior semicircular canal, ASC = superior semicircular canal, HSC = lateral semicircular canal, R = Right, L = Left semicircular canal plane, respectively. The medial cross represents the Y-axis and Z-axis. Figure 4 Solid model of human semicircular canal spatial attitude Red, blue, and green represent the normal vectors of the right lateral semicircular canal plane, posterior semicircular canal plane and superior semicircular canal plane, respectively. The medial cross represents the Y-axis and Z-axis.
https://openalex.org/W2903897489	http://www.scielo.br/pdf/rlae/v26/0104-1169-rlae-26-e2947.pdf	English	null	Labor conditions and the meanings of nursing work in Barcelona	Revista latino-americana de enfermagem	2,018	cc-by	5,522	1 PhD, Researcher, Corporación Sanitaria Parc Taulí -1, Hospital Universitario, Sabadell- Barcelona, Catalonia, Spain. Member of PETRO (Personas que Trabajan em Organizaciones, People Working in Organizations) research group. 2 PhD, Full Professor, Autonomous University of Barcelona (UAB), Psychology, Barcelona, Catalonia, Spain. San Buenaventura University (USB), Psychology, Cali, Colombia. Director of PETRO and WONPUM (Working Under New Public Management) research groups. 3 PhD, Professor, Politécnica del Litoral High School, ESPOL-ESPAE, Graduate School of Management, Campus Peñas Malecón 100 y Loja, P.O., Guayaquil, Box 09-01-5863, Ecuador. Member of PETRO and WONPUM research groups. Labor conditions and the meanings of nursing work in Barcelona Alberto Granero1 Josep M Blanch2 Paola Ochoa3 Alberto Granero1 Josep M Blanch2 Paola Ochoa3 Objective: to analyze the relationship between the quantitative assessment of working conditions and the qualitative perception of one’s own work experience. Method: a sample of 1,760 nursing professionals from Barcelona answered a questionnaire assessing their working conditions and summarized their own current work experience in five key words. Results: the textual corpus of the meanings of nursing work included 8043 lexical forms, which were categorized and codified. Respondents who rated their work conditions the highest expressed a vision of their work in terms of autonomy, achievement and well-being, while those who rated their work conditions the lowest talked mostly of exhaustion, depersonalization and negative climate. A correspondence analysis showed a close relationship between the quantitative assessments of working conditions and the verbal codes of the meaning of work. Conclusions: the meanings given to work were not only consistent with the numerical evaluations of the working conditions but also made them more understandable. The information obtained poses challenges for reflection and indicates ways to promote the positive aspects and prevent the negative conditions of nursing work. Descriptors: Nursing; Work; Occupational Health; Working Conditions; Workload; Quality of Life. How to cite this article Granero A, Blanch JM, Ochoa P. Labor conditions and the meanings of nursing work in Barcelona. Rev. Latino-Am. Enfermagem. 2018;26:e2947. [Access ___ __ ____]; Available in: ____________________. DOI: http://dx.doi. org/10.1590/1518-8345.2342.2947 URL day month year How to cite this article Granero A, Blanch JM, Ochoa P. Labor conditions and the meanings of nursing work in Barcelona. Rev. Latino-Am. Enfermagem. 2018;26:e2947. [Access ___ __ ____]; Available in: ____________________. DOI: http://dx.doi. org/10.1590/1518-8345.2342.2947 URL day month year Original Article Rev. Latino-Am. Enfermagem 2018;26:e2947 DOI: 10.1590/1518-8345.2342.2947 www.eerp.usp.br/rlae Original Article Rev. Latino-Am. Enfermagem 2018;26:e2947 DOI: 10.1590/1518-8345.2342.2947 www.eerp.usp.br/rlae Original Article Original Article Rev. Latino-Am. Enfermagem 2018;26:e2947 DOI: 10.1590/1518-8345.2342.2947 www.eerp.usp.br/rlae Labor conditions and the meanings of nursing work in Barcelona Introduction way for the second- and third-cycle training (Master’s and Doctorate) of nursing as an autonomous discipline and the development of the profession in the areas of assistance, teaching, research and management(24). In contrast to this positive trend, the conditions of application of this work potential evolved in the opposite direction in a series of organizational aspects. The initial situation was marked by a high ratio of patients per professional, well above the European average, which already entails a high care pressure. On this basis, a new organization and business management of health care work was implemented in recent decades, reinforced by certain policies to cope with the recent economic crisis. In this context, the imposition of measures to reduce the public deficit, especially the health budget, entailed reduced staffing levels and increased annual work time, posing obstacles to professionals’ careers and increasing psychosocial risks associated with burnout. This contemporary tension between the improvement of the professional qualifications and the worsening of the practice conditions of the profession has induced nursing professionals to reflect on the value, meaning and usefulness of their work. In this scenario, the objective of this research was to analyze the relationship between the quantitative assessment of working conditions and the qualitative perception of one’s own work experience in nursing professionals. The working conditions of health professionals, and particularly those of nursing professionals, have undergone profound changes in light of the general changes in the world of work. The scientific literature has analyzed the impact of working conditions on well- being and professional performance, as well as their multiple side effects on occupational health and the quality of the service provided. These effects appear as a negative spiral of care pressure, staff shortage, task overload, time deficit to execute everything and to do so well, distress and burnout, absenteeism and presentism, rotation and abandonment of the workplace and profession(1-11). The role of moderating variables in some of these effects, such as control of process and work content, horizontal and vertical social support, degree of adjustment between demands and labor resources, and the work-family balance or emotional load, has also been studied(12-15). Some research reports the economic and human cost of the lack of prevention of psychosocial risks in work in general and particularly in health care services(16-18). Introduction Another important topic of contemporary research in this field is the dynamics by which people give meaning and significance to their work in different sociocultural and organizational contexts and the role these cognitive processes play not only in shaping work experience but also in how professional practice is carried out(19-23). In this regard, psycho-sociological theories about the meaning of working argue that the meanings that people ascribe to their work do not derive only from immediate situations, contexts and conjunctures but also from a complex construction process involving values, ideals, goals, norms, rhetoric, strategies, beliefs, aspirations and sociocultural and personal expectations about work, profession and career. How to cite this article Granero A, Blanch JM, Ochoa P. Labor conditions and the meanings of nursing work in Barcelona. Rev. Latino-Am. Enfermagem. 2018;26:e2947. [Access ___ __ ____]; Available in: ____________________. DOI: http://dx.doi. org/10.1590/1518-8345.2342.2947 URL day month year Granero A, Blanch JM, Ochoa P. Labor conditions and the meanings of nursing work in Barcelona. Rev. Latino-Am. Enfermagem. 2018;26:e2947. [Access ___ __ ____]; Available in: ____________________. DOI: http://dx.doi. org/10.1590/1518-8345.2342.2947 URL day month year Rev. Latino-Am. Enfermagem 2018;26:e2947. 2 www.eerp.usp.br/rlae Results All the lexical forms were categorized and codified according to a dictionary constructed from a theoretical model according to which the meanings of the work are distributed along a bipolar semantic continuum. One of these slopes contains negative connotations of the work experience, such as malaise and dissatisfaction, as well as various aspects covered by the model of burnout, such as physical and emotional exhaustion, depersonalization, cynicism and inefficacy. On the other side are positive work meanings associated with well- being and satisfaction, realization and efficacy, as well as the components of the work engagement model (vigor, dedication and absorption)(21-23). The cCT is based on a theoretical model according to which the working conditions are structured around a triple relationship of the organization with the method, the environment and the person. The questionnaire evaluates the psychosocial components of working conditions (WC) that, depending on their form of presence and level of intensity, can function as protectors and promoters of health, well-being and quality of work life or as psychosocial risk factors. The cCT includes 44 closed items, presented in a Likert-type format of 11 ranks ranging from 0 (minimum value) to 10 (maximum value). The obtained psychometric data adequately reproduced the proposed theoretical model structure and showed a high internal consistency, with alpha values ranging from 0.852 to 0.983. In figure 1, the mean, standard deviation and Cronbach’s alpha of the entire questionnaire are specified, and each of the factors and scales are included. The mean valuation of WC, in scales ranging from 0-10, was 5.77 (SD=1.555), as shown in Figure 1. On the other hand, the textual corpus of the MW included 8043 lexical forms that, in turn, were found to be of the same order of magnitude; 62% had positive connotations (care, companionship, commitment, etc.) and the remaining 38% had negative connotations (exhaustion, malaise, overload, etc.). A correspondence analysis showed a close and statistically significant relationship between the quantitative assessments of working conditions and the verbal codes of meaning of the work (χ2 = 1434.01, p <0.001 and degrees of freedom = 93), as shown in Table 1. The mixed design of the research combined the qualitative technique of the analysis of textual content for the study of meanings expressed by keywords and the quantitative techniques of descriptive statistical analysis of the WCs and of factorial analysis of correspondences between WC and meaning of work (MW). Method Through the website of the Official College of Nurses of Barcelona (COIB) and some social networks of sector associations, 1,760 people, from a finite population of 32,463 nursing professionals, were accessed through simple random sampling. These individuals were working nurses in the province of Barcelona in June 2014. The size of this sample provided a confidence level of 99% and a confidence interval of 3%. The characteristic profile of the participants (which reproduced with remarkable precision that of the reference population) was that of women (85.9%), 41.9 years old (standard deviation SD = 10.4), with 18.3 years (SD = 10.8) of seniority since graduation and 13.5 years (SD= 10.0) in the workplace, with a general education (95.5%), stable or permanent contract (73.0%), full-time employment (76.1%), day shift (80.5%), in the health care industry (81.0%), hospital or clinic (53.3%), public (32.7%) or mixed ownership (57.7%). All of them voluntarily answered a survey that collected information on the central variables of the study by including the Questionnaire on In recent decades, the development of the nursing profession has followed a paradoxical development at both the global and local levels: on the one hand, training in skills and material and technological resources for professional performance has improved, while on the other, the conditions of work have become more hard, complex and difficult. This is precisely what happened in the environment of Barcelona, where, over the last half-century, the profession went from basic training as a Technical Medical Assistant (1953-1979), through the Diploma in Nursing (1977-2012) to the Graduate degree in Nursing (begun in 2009), which opened the 3 Granero A, Blanch JM, Ochoa P. Granero A, Blanch JM, Ochoa P. answers, commitment of return of results and scientific and responsible use of the information were applied. Working Conditions(25) (Cuestionario de Condiciones de Trabajo, cCT), composed of sets of closed items with a Likert-type format with ranges from 0 to 10 and an open question regarding the meaning of the work that invited respondents to summarize the actual work experience itself in 5 “keywords.” The lexical forms obtained from the answers were later categorized and codified. Finally, the respondents filled out a section of sociodemographic and work data. www.eerp.usp.br/rlae Results For the correspondence analysis, the WC-related scores were divided into four categories: “very bad” WC (X = 0-2.5), “bad” WC (X = 2.5-5), “good” WC (X = 5- 7.5) and “very good” WC (X = 7.5-10). In the descriptive statistical analysis, the quantitative variables were presented in terms of mean (X) and standard deviation (SD), while the qualitative variables were in the form of frequencies and percentages. In the factorial analysis of correspondences, the chi square (χ²) was used to determine the relationships between the WC and MW variables from the data in the contingency table. MW code groupings were observed according to the WC valuation levels. As shown in Figure 2, at the level of the “Very Good” WCs corresponded the following codes of meaning: autonomy, achievement and well-being. The “Good” WCs were associated with satisfaction, opportunities, good social relations, vigor, effectiveness, good socioeconomic conditions, commitment, responsibility, competence, ethics, care, economic recognition and general recognition. The “Bad” WCs corresponded to the elements of exhaustion, inappropriate work, negative climate, disorganization, overload, poor socioeconomic conditions, little recognition, malaise, injustice, organizational cynicism, dissatisfaction, mismanagement, scarce resources and inefficacy. Finally, the level of “Very Bad” WCs corresponded with depersonalization. The project of this research was approved by the Bioethics Commission of the University of Barcelona. In its development, the international rules regarding informed consent, confidentiality of data of participants and institutions, safeguarding the anonymity of the In Table 2, this distribution of the ST codes is reflected according to the levels of assessment of the CT. 4 Rev. Latino-Am. Enfermagem 2018;26:e2947. Rev. Latino-Am. Enfermagem 2018;26:e2947. Rev. Latino-Am. Enfermagem 2018;26:e2947. g ; Figure 1 - Theoretical Model of cCT: Cronbach’s Alpha (α), Mean (X) and Standard Deviation (SD) of the Questionnaire, Factors and Scales, Barcelona, CA, Spain, 2014-2015 Figure 1 - Theoretical Model of cCT: Cronbach’s Alpha (α), Mean (X) and Standard Deviation (SD) of the Questionnaire, Factors and Scales, Barcelona, CA, Spain, 2014-2015 Table 1- Correspondence analysis between evaluation levels of working conditions and codes of meaning of work. Barcelona, CA, Spain, 2014-2015. Table 1- Correspondence analysis between evaluation levels of working conditions and codes of mean Barcelona, CA, Spain, 2014-2015. Table 1- Correspondence analysis between evaluation levels of working conditions and codes of meaning of work. Barcelona, CA, Spain, 2014-2015. www.eerp.usp.br/rlae Results Granero A, Blanch JM, Ochoa P. Figure 2 - Conditions and Meanings of Work. Barcelona, CA, Spain, 2014-2015 Figure 2 - Conditions and Meanings of Work. Barcelona, CA, Spain, 2014-2015 Figure 2 - Conditions and Meanings of Work. Barcelona, CA, Spain, 2014-2015 Tabela 2 - Distribuição dos códigos de Significados do Trabalho (ST) segundo níveis de avaliação das Condições de Trabalho (NACT). Barcelona, CA, Espanha, 2014-2015. Tabela 2 - Distribuição dos códigos de Significados do Trabalho (ST) segundo níveis de avaliação das Condições de Trabalho (NACT). Barcelona, CA, Espanha, 2014-2015. Tabela 2 - Distribuição dos códigos de Significados do Trabalho (ST) segundo níveis de avaliação das Condições de Trabalho (NACT). Barcelona, CA, Espanha, 2014-2015. Tabela 2 - Distribuição dos códigos de Significados do Trabalho (ST) segundo níveis de avaliação das Condições de Trabalho (NACT). Barcelona, CA, Espanha, 2014-2015. Results Meaning of Work Mass Score in dimension Inertia Contribution From the points to the inertia of the dimension From the dimension to the inertia of the point 1 2 1 2 1 2 Total WC Very Bad 0,027 -1,340 -1,521 0,026 0,122 0,715 0,754 0,213 0,967 WC Bad 0,280 -0,843 0,257 0,082 0,491 0,208 0,978 0,020 0,998 WC Good 0,575 0,273 -0,084 0,019 0,106 0,046 0,892 0,018 0,911 WC Very Good 0,118 0,984 0,154 0,050 0,281 0,031 0,923 0,005 0,927 Total 1,000 0,178 1,000 1,000 Overload 0,057 -0,268 -0,199 0,002 0,010 0,025 0,784 0,095 0,879 Bad management 0,014 -1,214 -0,237 0,009 0,051 0,009 0,974 0,008 0,982 Disorganization tasks 0,012 -0,887 0,583 0,004 0,022 0,044 0,905 0,086 0,991 Negative Climate 0,031 -0,798 0,462 0,009 0,049 0,074 0,930 0,068 0,998 Injustice 0,014 -1,187 0,513 0,009 0,050 0,042 0,958 0,039 0,997 Inappropriate work 0,015 -0,585 0,439 0,002 0,013 0,033 0,887 0,109 0,996 Lack of Resources 0,004 -0,763 -0,819 0,001 0,006 0,030 0,787 0,199 0,986 Poor socioeconomic Conditions 0,063 -0,523 -0,397 0,008 0,043 0,112 0,853 0,108 0,961 Dissatisfaction 0,017 -1,214 0,002 0,011 0,063 0,000 0,978 0,000 0,978 Discomfort 0,060 -0,989 -0,361 0,025 0,144 0,088 0,960 0,028 0,988 Exhaustion 0,070 -0,499 0,106 0,008 0,043 0,009 0,912 0,009 0,921 Organizational Cynicism 0,013 -1,185 0,293 0,008 0,045 0,013 0,959 0,013 0,972 Depersonalization 0,005 -1,538 -1,665 0,007 0,029 0,155 0,722 0,186 0,908 Inefficacy 0,006 -1,425 1,624 0,006 0,029 0,173 0,762 0,217 0,979 Good socioeconomic Conditions 0,019 0,498 0,021 0,002 0,012 0,000 0,941 0,000 0,942 Opportunities 0,043 0,593 0,000 0,006 0,037 0,000 1,000 0,000 1,000 Satisfaction 0,035 0,597 0,064 0,006 0,031 0,002 0,894 0,002 0,896 Wellbeing 0,049 0,690 0,033 0,010 0,057 0,001 0,973 0,000 0,974 Vigor 0,037 0,559 0,146 0,005 0,028 0,009 0,917 0,014 0,931 Commitment 0,052 0,580 0,040 0,007 0,044 0,001 0,985 0,001 0,986 Ethics 0,017 0,303 0,046 0,001 0,004 0,000 0,728 0,004 0,732 Good social Relations 0,055 0,450 0,040 0,005 0,028 0,001 0,979 0,002 0,980 Realization 0,014 0,789 -0,052 0,004 0,021 0,000 0,999 0,001 1,000 Efficacy 0,069 0,523 -0,057 0,008 0,046 0,002 0,992 0,003 0,994 Competences 0,009 0,303 -0,046 0,001 0,002 0,000 0,640 0,003 0,643 Care 0,135 0,274 -0,104 0,004 0,025 0,016 0,925 0,029 0,955 Responsibility 0,045 0,397 0,045 0,003 0,017 0,001 0,887 0,003 0,890 Autonomy 0,012 0,875 0,173 0,004 0,023 0,004 0,883 0,008 0,891 Little Recognition 0,014 -0,868 0,863 0,005 0,026 0,118 0,803 0,174 0,977 Recognition 0,007 0,277 0,668 0,001 0,001 0,036 0,308 0,392 0,700 Economic Recognition 0,008 0,069 0,044 0,000 0,000 0,000 0,127 0,011 0,138 Total 1,000 0,178 1,000 1,000 www.eerp.usp.br/rlae 5 Granero A, Blanch JM, Ochoa P. www.eerp.usp.br/rlae † n: frequency of meaning of work codes according to levels of assessment of working conditions; Discussion While the numbers presented indicate the relative levels of satisfaction for the conditions of nursing work in the social and organizational environment studied, the codes of meaning associated with them give names to the aspects of those conditions that need to be strengthened and those that need to be modified. The results of this research are in general agreement with the information provided by the literature on the work experience and quality of working life for nursing professionals in the new environment resulting from the successive contemporary reforms of the health system. However, the results include some content that points beyond what is already known. First, the results allow for synthesizing, condensing and qualifying the effects of the political, managerial and technological innovations observed in the most diverse health contexts. Second, the results show how these new organizational demands impact nurses psychologically and how they are approached psychologically in a doubly critical scenario: a reform of health services accelerated and intensified by strong financial constraints, which translates into strategies to cut staff and increase the ratio of patients per professional, thereby increasing pressure on health care by a quantitative and qualitative over demand of care and cognitive and emotional work. Overall, the study contains some notable weaknesses and strengths. Among its methodological limitations is the use of self-report measures within the framework of a cross-sectional design, which in turn did not allow for causal inferences. However, none of these characteristics hampered the achievement of the research goal concerning the relationship between numerical scores and verbal responses referring to the same phenomena of experience of working conditions. However, the fact that fieldwork was developed in the context of a crisis in the Spanish health system’s financing, which had a significant impact on working conditions in nursing in Barcelona, raises reasonable doubts as to whether the results presented would have been different if they had been obtained before or after this critical period. Among the strengths of the research, we highlight the breadth of the thematic field covered and its mixed design, which combined the use of numerous scales composed of a series of closed items for the collection of quantitative data, with an open question as a source of the qualitative data. This allowed the implementation of various techniques for studying the information obtained, such as statistical analysis, thematic content and correspondences. Results Rev. Latino-Am. Enfermagem 2018;26:e2947. 6 Discussion The agenda for future studies linked to the present includes the challenge of improving understanding of the mechanisms involved in an observed paradox: the nursing professionals who participated in the research were generally highly affected by a life care practice experienced as very stressful, exhausting and fatiguing while they maintained a positive vision of it, a high professional self-esteem and a strong involvement in the work. Overall, the study contains some notable weaknesses and strengths. Among its methodological limitations is the use of self-report measures within the framework of a cross-sectional design, which in turn did not allow for causal inferences. However, none of these characteristics hampered the achievement of the research goal concerning the relationship between numerical scores and verbal responses referring to the same phenomena of experience of working conditions. As a whole, the information provided by the study revolves around three main axes. First, the participants show a high coherence between their numerical assessment of their own working conditions and their textual characterization of the meanings of their work experience. Second, they think of their profession in terms of a caring relationship and perceive their work and professional experience in relatively positive terms, indicating aspects to be protected and strengthened. Third, they also report important shortcomings, deficiencies and dysfunctions in the design, organization and management of this work and the conditions of that professional exercise. This negative aspect is especially marked by work overload and concepts associated with it, such as the lack of time and human resources available to deal effectively with organizational demands. Other highlights are a perceived lack of autonomy in work as well as opportunities for professional development. The moderately positive assessment of the conditions for the realization of one’s own work experience is in line with what has been gathered by current reports on the quality of work life within the general context of contemporary working conditions(17) and also on what specifically concerns the provision of public services to persons(26-27) and, within this group, those of the health sector in particular(1,4,21-23) and specifically those in the field of nursing(2- 3,13,18,28-31). The vision provided by the participants is also consistent with what the literature reports regarding the dark side of the characteristics and tendencies of working conditions in general, both for their psychosocial risk factors(16-18) and because of the lack of wellbeing in health care work(5-7,10-12,26-27,30-31). Results Work Meaning Codes Levels of valuation of working conditions (LVWC) Very Bad Bad Good Very Good Accumulated n† % n† % n† % n† % N‡ % Overload 19 4.2 148 32.4 260 56.9 30 6.6 457 5.7 Bad management 10 8.9 63 55.8 40 35.4 0 0.0 113 1.4 Disorganization Tasks 3 3.2 49 52.7 40 43.0 1 1.1 93 1.2 Negative Climate 9 3.6 124 50.0 108 43.5 7 2.8 248 3.1 Injustice 6 5.2 69 60.0 40 34.8 0 0.0 115 1.4 Inappropriate work 4 3.2 56 45.2 56 45.2 8 6.5 124 1.5 Lack of Resources 3 9.4 13 40.6 15 46.9 1 3.1 32 0.4 Poor socioeconomic conditions 30 5.9 188 36.9 275 54.0 16 3.1 509 6.3 Dissatisfaction 11 7.9 80 57.6 47 33.8 1 0.7 139 1.7 Discomfort 40 8.3 237 49.3 197 41.0 7 1.5 481 6.0 Exhaustion 20 3.6 223 39.8 296 52.9 21 3.8 560 7.0 Organizational Cynicism 7 6.7 62 59.0 34 32.4 2 1.9 105 1.3 Depersonalization 7 17.5 22 55.0 11 27.5 0 0.0 40 0.5 Inefficacy 1 2.1 35 74.5 10 21.3 1 2.1 47 0.6 Good socioeconomic Conditions 2 1.3 26 17.0 95 62.1 30 19.6 153 1.9 Opportunities 2 0.6 48 14.0 228 66.7 64 18.7 342 4.3 Satisfaction 3 1.1 43 15.4 172 61.4 62 22.1 280 3.5 Wellbeing 2 0.5 49 12.5 256 65.3 85 21.7 392 4.9 Vigor 2 0.7 49 16.6 182 61.7 62 21.0 295 3.7 Commitment 3 0.7 63 14.9 273 64.7 83 19.7 422 5.2 Ethics 1 0.7 28 20.3 92 66.7 17 12.3 138 1.7 Good social Relations 3 0.7 77 17.3 293 66.0 71 16.0 444 5.5 Realization 0 0.0 10 8.9 79 70.5 23 20.5 112 1.4 Efficacy 5 0.9 83 15.1 369 67.0 94 17.1 551 6.9 Competences 2 2.7 16 21.6 42 56.8 14 18.9 74 0.9 Care 21 1.9 222 20.5 692 63.8 149 13.7 1084 13.5 Responsibility 2 0.6 66 18.3 242 67.0 51 14.1 361 4.5 Autonomy 0 0.0 10 10.1 62 62.6 27 27.3 99 1.2 Little Recognition 2 1.8 61 54.0 49 43.4 1 0.9 113 1.4 Recognition 0 0.0 16 27.6 30 51.7 12 20.7 58 0.7 Economic Recognition 1 1.6 16 25.8 39 62.9 6 9.7 62 0.8 Frequencies (n) and Percentages (%) 221 2.7 2252 28.0 4624 57.5 946 11.8 8043 100 LVWC: Levels of valuation of Working Conditions † n: frequency of meaning of work codes according to levels of assessment of working conditions; ‡ N: accumulated frequency by codes of meaning of work. www.eerp.usp.br/rlae Conclusion Work-to- family conflict as a mediator of the relationship between job satisfaction and turnover intention. J Adv Nurs. 2015; 71(10):2350-63. doi: 10.1111/jan.12706 Referencias 1. Aiken LH, Sloane DM, Clarke S, Poghosyan L, Cho E, You L. Importance of work environments on hospital outcomes in nine countries. Qual Health Res. 2011;23(4):357-64. doi: 10.1093/intqhc/mzr022 2. Aiken LH, Sermeus W, Van Den Heede K, Sloane DM, Busse R, Mckee M, et al. Patient safety, satisfaction, and quality of hospital care: cross sectional surveys of nurses and patients in 12 countries in Europe and the United States. Brit Med J. 2012;344:1717. doi: 10.1136/bmj. e1717 1. Aiken LH, Sloane DM, Clarke S, Poghosyan L, Cho E, You L. Importance of work environments on hospital outcomes in nine countries. Qual Health Res. 2011;23(4):357-64. doi: 10.1093/intqhc/mzr022 13. Cortese C, Colombo L, Ghislieri C. Determinants of nurses’ job satisfaction: the role of work-family conflict, job demand, emotional charge and social support. J Nurs Manag. 2010;18:35-43. doi: 10.1111/j.1365- 2834.2009.01064.x 2. Aiken LH, Sermeus W, Van Den Heede K, Sloane DM, Busse R, Mckee M, et al. Patient safety, satisfaction, and quality of hospital care: cross sectional surveys of nurses and patients in 12 countries in Europe and the United States. Brit Med J. 2012;344:1717. doi: 10.1136/bmj. e1717 14. Jourdain G, Chênevert D. Job-demands-resources, burnout and intention to leave the nursing profession: A questionnaire survey. Int J Nurs Stud. 2010;47:709- 22. doi: 10.1016/j.ijnurstu.2009.11.007 15. Yamaguchi Y, Inoue T, Harada H, Oike M. Job control, work-family balance and nurses’ intention to leave their profession and organization: A comparative cross-sectional survey. Int J Nurs Stud. 2016;64:52-62. doi: 10.1016/j.ijnurstu.2016.09.003 3. Aiken LH, Sloane DM, Bruyneel L, Van den Heede K, Sermeus W. Nurses’ reports of working conditions and hospital quality of care in 12 countries in Europe. Int J Nurs Stud. 2013;50:143–53. doi: 10.1016/j. ijnurstu.2012.11.009 16. EU-OSHA. Calculating the Costs of Work-Related Stress and Psychosocial Risks [Internet]. Luxembourg: European Agency for Safety and Health at Work. Publications Office of the European Union; 2014. [cited June 18, 2017]. Available from: https://osha.europa. eu/sites/default/files/publications/documents/en/ publications/literature_reviews/calculating-the-cost- of-work-related-stress-and-psychosocial-risks/cost-of- work-related-stress.pdf 4. Blanch JM. Quality of working life in mercantilized hospitals and universities. Pap Psicol. 2014;35(1):40-7. doi: 10.1080 / 15379418 5. Chan ZCY, Tam WS, Lung MKY, Wong WY, Chau CW. A systematic literature review of nurse shortage and the intention to leave. J Nurs Manag. 2013;21(4):605-13. doi: 10.1111/j.1365-2834.2012.01437.x 6. Faller MS, Gates MG, Georges JM, Connelly CD. Work-related burnout, job satisfaction, intent to leave, and nurse-assessed quality of care among travel nurses. J Nurs Adm. 2011;41(2):71-7. doi: 10.1097/ NNA.0b013e3182059492 17. www.eerp.usp.br/rlae Conclusion The eight thousand key words with which the respondents summarized their work experience in their current organizational context agreed with their numerical assessments of the conditions of their professional practice, giving such scores a more precise meaning. In this respect, the contribution of this study has a twofold aspect: on the one hand, it ratifies in the current Barcelona environment quantitative aspects already known regarding the real conditions of the contemporary practice of health care. On the other, it involves progress in and deepening of the qualitative 7 7 Granero A, Blanch JM, Ochoa P. knowledge of the investigated reality by specifying the positive and negative nuances of current nursing work experience with words, categories and semantic codes. 9. Nei D, Snyder LA, Litwiller BJ. Promoting retention of nurses: a metaanalytic examination of causes of nurse turnover. Health Care Manage R. 2014; 40(3)237–53. doi: 10.1097/HMR.0000000000000025 By showing the image of an ambivalent work experience characterized by the tension between the vocation to care and the imposition of a chronic task overload, professionals have noted challenges for reflection and paths for policies committed to a double mission: strengthening the positive aspects and correcting the negative aspects of the nursing staff’s work conditions, with a view to improving their occupational health and well-being while optimizing the quality of the service they provide to their patients and their community. 10. Van Bogaert P, Clarke S, Roelant E, Meulemans H, Van De Heyning P. Impacts of unit level nurse practice environment and burnout on nurse-reported outcomes: a multilevel modelling approach. J Clin Nurs. 2010; 50(3):357-65. doi: 10.1111/j.1365-2702.2009.03128.x 11. Van der Doef M, Bannink F, Verhoeven C. Job condi tions, job satisfaction, somatic complaints and burnout among East African nurses. J Clin Nurs. 2012;21(11- 12):1763-75. doi: 10.1111/j.1365-2702.2011.03995.x 12. Chen IH, Brown R, Bowers BJ, Chang W-Y. Work-to- family conflict as a mediator of the relationship between job satisfaction and turnover intention. J Adv Nurs. 2015; 71(10):2350-63. doi: 10.1111/jan.12706 10. Van Bogaert P, Clarke S, Roelant E, Meulemans H, Van De Heyning P. Impacts of unit level nurse practice environment and burnout on nurse-reported outcomes: a multilevel modelling approach. J Clin Nurs. 2010; 50(3):357-65. doi: 10.1111/j.1365-2702.2009.03128.x 11. Van der Doef M, Bannink F, Verhoeven C. Job condi tions, job satisfaction, somatic complaints and burnout among East African nurses. J Clin Nurs. 2012;21(11- 12):1763-75. doi: 10.1111/j.1365-2702.2011.03995.x 12. Chen IH, Brown R, Bowers BJ, Chang W-Y. Referencias EUROFOUND (European Foundation for the Improvement of Living and Working Conditions). Sixth European Working Conditions Survey – Overview report. Luxembourg: Publications Office of the European Union; 2016. Available at: http://www.eurofound.europa.eu/ publications/report/2016/working-conditions/sixth- european-working-conditions-survey-overview-report 18. Ceballos-Vásquez P, Rolo-González G, Hérnandez- Fernaud E, Díaz-Cabrera, D, Paravic-Klijn, T, Burgos- Moreno, M. Psychosocial factors and mental work load: a reality perceived by nurses in intensive care units. Rev. Latino-Am. Enfermagem. 2015;23(2):315-22. doi:10.1590/0104-1169.0044.2557. 7. Humphries N, Morgan K, Conry MC, McGowan Y, Montgomery A, McGee H. Quality of care and health professional burnout: narrative literature review. Int J Qual Health Care. 2014;27(4):293-307. doi: 10.1108/ IJHCQA-08-2012-0087 8. Kuhlmann E, Annandale E. Researching transfor mations in healthcare services and policy in inter national perspective: An introduction. Cur Sociol. 2012;60(4):401-14. doi: 10.1177/0011392112438325 8 8 Rev. Latino-Am. Enfermagem 2018;26:e2947. magem. 2011;19(2):340-7. doi: 10.1590/S0104- 11692011000200016 19. Ardichvili A, Kuchinke KP. International Perspectives on the Meanings of Work and Working: Current Research and Theory. Adv Develop Hum Resour. 2009;11(2):155- 67. doi: 10.1177/1523422309333494 19. Ardichvili A, Kuchinke KP. International Perspectives on the Meanings of Work and Working: Current Research and Theory. Adv Develop Hum Resour. 2009;11(2):155- 67. doi: 10.1177/1523422309333494 29. Lu H, Barriball KL, Zhang X, While AE. Job satisfaction among hospital nurses revisited: A systematic review. Int J Nurs Stud. 2012;49(8):1017-38. doi: 10.1016/j. ijnurstu.2011.11.009 20. Arnoux C, Sovet L, Lhotellier L, Di Fabio A, Bernaud JL. Perceived Work Conditions and Turnover Intentions: The Mediating Role of Meaning of Work. Front Psychol. 2016:7:1-9. doi: 10.3389/fpsyg.2016.00704 21. Guido L, Goulart C, Silva R, Lopes L, Ferreira E. Stress and Burnout among multidisciplinary residents. Rev. Lati no-Am. Enfermagem. 2012;20(6):1064-71. doi: http:// dx.doi.org/10.1590/S0104-11692012000600008. 30. Chan ZC, Tam WS, Lung MK, Wong WY, Chau CW. A systematic literature review of nurse shortage and the intention to leave. J Nurs Manag. 2013;21:605–13. doi: 10.1111/j.1365-2834.2012.01437.x 31. Zhang LM, You LM, Liu Ke, Zheng J, Fang JB, Lu MM, et al. The association of Chinese hospital work environment with nurse Burnout, job satisfaction, and intention to leave. Nurs Outlook. 2014;62(2):128-37. doi: 10.1016/j.outlook.2013.10.010 22. Ochoa P, Blanch JM. Work, malaise and wellbe ing in Spanish and Latin American doctors. Rev Saúde Pública. 2016;50(21):1-14. doi: 10.1590/S1518- 8787.2016050005600 23. Blanch JM, Ochoa PJ, Sahagún MA. Resignification of work and the medical profession under the new health management. In: Ansoleaga E, Artaza O, Suárez J, editors. Persons Caring for People: Human Dimension and Health Work. [Internet]. Copyright © 2018 Revista Latino-Americana de Enfermagem This is an Open Access article distributed under the terms of the Creative Commons (CC BY). This license lets others distribute, remix, tweak, and build upon your work, even commercially, as long as they credit you for the original creation. This is the most accommodating of licenses offered. Recommended for maximum dissemination and use of licensed materials. www.eerp.usp.br/rlae Received: Jan. 18th 2017 Accepted: Aug. 5th 2017 Referencias Santiago de Chile: PAHO / WHO - Pan American Health Organization / World Health Organization; 2012. p. 165-75. Available from: https://www.researchgate.net/publication/287997653_ Resignificacion_del_trabajo_y_de_la_profesion_ medica_bajo_la_nueva_gestion_sanitaria 24. Council of Catalonia Nurses. Training, Teaching and Research in Mental Health Catalan Nurses [Internet]. Barcelona: Council of Catalonia Nurses; 2016. [cited June 30, 2017]. Available from: https://www.agoraden fermeria.eu/files/news/crai_es.pdf 25. Blanch JM, Sahagun M, Cervantes G. Factor structure of Working Conditions Scale. J Work Organ Psychol. 2010;26(3):175-89. doi: 10.5093/tr2010v26n3a2 25. Blanch JM, Sahagun M, Cervantes G. Factor structure of Working Conditions Scale. J Work Organ Psychol. 2010;26(3):175-89. doi: 10.5093/tr2010v26n3a2 25. Blanch JM, Sahagun M, Cervantes G. Factor structure of Working Conditions Scale. J Work Organ Psychol. 2010;26(3):175-89. doi: 10.5093/tr2010v26n3a2 26. Tummers LG, Bekkers V, Vink E, Musheno M. Coping during public service delivery: A conceptualization and systematic review of the literature. J Public Adm Res Theory. 2015;25(4):1099-126. doi: 10.1093/jopart/ muu056 26. Tummers LG, Bekkers V, Vink E, Musheno M. Coping during public service delivery: A conceptualization and systematic review of the literature. J Public Adm Res Theory. 2015;25(4):1099-126. doi: 10.1093/jopart/ muu056 27. Tummers LG, Bekkers V, Steijn AJ. Policy alienation of public professionals: Application in a new public management context. Pub Manag Rev. 2009;11(5):685- 706. doi: 10.1080/14719030902798230 28. Mininel V, Baptista P, Felli V. Psychic workloads and strain processes in nursing workers of Brazil ian university hospitals. Rev. Latino-Am. Enfer Received: Jan. 18th 2017 Accepted: Aug. 5th 2017 Corresponding Author: Josep M Blanch UAB Universitat Autònoma de Barcelona Faculty of Psychology Edificio B, Campus UAB Bairro: Bellaterra-Barcelona CEP: 08193, Catalonia, Spain E-mail: jmbr.blanch@gmail.com This license lets others distribute, remix, tweak, and build upon your work, even commercially, as long as they credit you for the original creation. This is the most accommodating of licenses offered. Recommended for maximum dissemination and use of licensed materials. www.eerp.usp.br/rlae
https://openalex.org/W2575385740	https://dash.harvard.edu/bitstream/1/34375077/1/5517355.pdf	English	null	A taxonomy of medical uncertainties in clinical genome sequencing	Genetics in medicine	2,017	cc-by	6,916	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 Permanent link http://nrs.harvard.edu/urn-3:HUL.InstRepos:34375077 Citation Han, Paul K. J., Kendall L. Umstead, Barbara A. Bernhardt, Robert C. Green, Steven Joffe, Barbara Koenig, Ian Krantz, Leo B. Waterston, Leslie G. Biesecker, and Barbara B. Biesecker. 2017. “A Taxonomy of Medical Uncertainties in Clinical Genome Sequencing.” Genetics in medicine : official journal of the American College of Medical Genetics 19 (8): 918-925. doi:10.1038/gim.2016.212. http://dx.doi.org/10.1038/gim.2016.212. Published Version doi:10.1038/gim.2016.212 Published Version doi:10.1038/gim.2016.212 Share Your Story The Harvard community has made this article openly available. Please share how this access benefits you. Submit a story . A Taxonomy of Medical Uncertainties in Clinical Genome Sequencing Citation Han, Paul K. J., Kendall L. Umstead, Barbara A. Bernhardt, Robert C. Green, Steven Joffe, Barbara Koenig, Ian Krantz, Leo B. Waterston, Leslie G. Biesecker, and Barbara B. Biesecker. 2017. “A Taxonomy of Medical Uncertainties in Clinical Genome Sequencing.” Genetics in medicine : official journal of the American College of Medical Genetics 19 (8): 918-925. doi:10.1038/gim.2016.212. http://dx.doi.org/10.1038/gim.2016.212. A Taxonomy of Medical Uncertainties in Clinical Genome Sequencing Citation Han, Paul K. J., Kendall L. Umstead, Barbara A. Bernhardt, Robert C. Green, Steven Joffe, Barbara Koenig, Ian Krantz, Leo B. Waterston, Leslie G. Biesecker, and Barbara B. Biesecker. 2017. “A Taxonomy of Medical Uncertainties in Clinical Genome Sequencing.” Genetics in medicine : official journal of the American College of Medical Genetics 19 (8): 918-925. doi:10.1038/gim.2016.212. http://dx.doi.org/10.1038/gim.2016.212. A Taxonomy of Medical Uncertainties in Clinical Genome Sequencing Paul K. J. Han, MD, MPH1, Kendall L. Umstead, MS2, Barbara A. Bernhardt, MS3, Robert C. Green, MD, MPH4,5,6,7, Steven Joffe, MD, MPH8, Barbara Koenig, PhD9, Ian Krantz, MD10, Leo B. Waterston, MA1, Leslie G. Biesecker, MD11, and Barbara B. Biesecker, PhD2 1Center for Outcomes Research and Evaluation, Maine Medical Center Research Institute, Portland, ME, USA Paul K. J. Han, MD, MPH1, Kendall L. Umstead, MS2, Barbara A. Bernhardt, MS3, Robert C. Green, MD, MPH4,5,6,7, Steven Joffe, MD, MPH8, Barbara Koenig, PhD9, Ian Krantz, MD10, Leo B. Waterston, MA1, Leslie G. Biesecker, MD11, and Barbara B. Biesecker, PhD2 1Center for Outcomes Research and Evaluation, Maine Medical Center Research Institute, Portland, ME, USA Author Manuscript 2Social and Behavioral Research Branch, National Human Genome Research Institute, NIH, Bethesda, MD, USA 3Division of Translational Medicine and Human Genetics, Perelman School of Medicine, 3Division of Translational Medicine and Human Genetics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA 4Division of Genetics, Department of Medicine, Brigham and Women’s Hospital, Boston, MA, USA 5Personalized Medicine, Partners Healthcare, Cambridge, MA, USA 6Department of Medicine, Harvard Medical School, Boston, MA, USA 7Broad Institute of MIT and Harvard, Boston, MA, USA Author Manuscript 8Department of Medical Ethics and Health Policy, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA 9Institute for Health & Aging, University of California, San Francisco, CA, USA 10Division of Human Genetics, The Children’s Hospital of Philadelphia, Philadelphia, PA, USA 11Medical Genomics and Metabolic Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, USA HHS Public Access Author Manuscript Published in final edited form as: Published in final edited form as: Genet Med. 2017 August ; 19(8): 918–925. doi:10.1038/gim.2016.212. Genet Med. 2017 August ; 19(8): 918–925. doi:10.1038/gim.2016.212. CONFLICTS OF INTEREST P.K.J.H., K.L.U., B.A.B., S.J., B.K., I.K., L.B.W., and B.B.B. have no conflicts to disclose. L.G.B. receives royalties from Genentech Corp., is an unpaid advisor to Illumina Corp., and receives honoraria from Wiley-Blackwell Inc. R.C.G. has received compensation for speaking or for advisory services from Illumina, Prudential, Helix, AIA, Invitae, and Roche. To whom correspondence should be addressed: Barbara B. Biesecker, Ph.D., barbara.biesecker@nih.gov, 31 Center Drive, Building 31, Room B1B36, Bethesda, MD 20892-2073. Accessibility Accessibility CONFLICTS OF INTEREST Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic subject always to the full Conditions of use:http://www.nature.com/authors/editorial_policies/license.html#terms Keywords uncertainty; genome sequencing; exome sequencing; next-generation sequencing; taxonomy Genet Med. Author manuscript; available in PMC 2017 August 07. Abstract Purpose—Clinical next generation sequencing (CNGS) is introducing new opportunities and challenges into the practice of medicine. Simultaneously, these technologies are generating uncertainties of unprecedented scale that laboratories, clinicians, and patients are required to Author Manuscript Author Manuscript Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:http://www.nature.com/authors/editorial_policies/license.html#terms CON C S O N S P.K.J.H., K.L.U., B.A.B., S.J., B.K., I.K., L.B.W., and B.B.B. have no conflicts to disclose. L.G.B. receives royalties from Genentech Corp., is an unpaid advisor to Illumina Corp., and receives honoraria from Wiley-Blackwell Inc. R.C.G. has received compensation for speaking or for advisory services from Illumina, Prudential, Helix, AIA, Invitae, and Roche. Page 2 Han et al. address and manage. We describe in this report the conceptual design of a new taxonomy of uncertainties around the use of CNGS in health care. Author Manuscript Methods—Interviews to delineate the dimensions of uncertainty in CNGS were conducted with genomics experts, and themes were extracted in order to expand upon a previously published three-dimensional taxonomy of medical uncertainty. In parallel we developed an interactive website to disseminate the CNGS taxonomy to researchers and engage them in its continued refinement. Results—The proposed taxonomy divides uncertainty along three axes: source, issue, and locus, and further discriminates the uncertainties into five layers with multiple domains. Using a hypothetical clinical example, we illustrate how the taxonomy can be applied to findings from CNGS and used to guide stakeholders through interpretation and implementation of variant results. Conclusion—The utility of the proposed taxonomy lies in promoting consistency in describing dimensions of uncertainty in publications and presentations, to facilitate research design and management of the uncertainties inherent in the implementation of CNGS. Author Manuscript INTRODUCTION The arrival of clinical next generation sequencing (CNGS) introduces new opportunities and challenges. It offers increased diagnostic precision and improved understanding of the causes and outcomes of an array of diseases at the individual level, and promises to advance predictive and preventive medicine on an unprecedented scale. Genomic technologies have already begun to deliver on this promise. Although these technologies have been available for only a few years, their integration into clinical practice for the evaluation of patients with a variety of disorders has been swift and their demonstrated utility impressive.1,2 Genomic testing has successfully advanced the diagnosis of previously undiagnosed rare disorders, identification of high risk of disease among healthy individuals, and development of targeted cancer treatments. Author Manuscript Although medicine is replete with uncertainties, genomic technologies introduce uncertainties of unique scale and type.1,3 Genomic testing is technically complex, involving multiple steps—from sample acquisition to clinical report generation to communication of results to the patient—each of which introduces uncertainty regarding, for example, the accuracy and reliability of test results. The clinical uses of genomic testing introduce other uncertainties regarding the benefits and harms of genomic test information, the optimal strategies for communicating this information to patients, and the consequences of genomic testing for patients, family members, the health care system, and society. Author Manuscript Whether CNGS is being used for diagnosis, predispositional testing, preconception screening, prenatal screening, pharmocogenomic profiling, or some other purpose, the introduction of CNGS into the clinic requires laboratories, clinicians, patients, and families to address and manage the many uncertainties it raises. Yet, the first step in this endeavor is Han et al. Page 3 to distinguish and understand these specific uncertainties and the extent to which they are reducible by the acquisition of further knowledge. This, in turn, enables thoughtful consideration of their practical and ethical implications. Clinicians can then help patients appreciate the uncertainties pertaining to their testing experience and draw on personal resources to cope with them. Author Manuscript Author M Author Manuscript Cataloguing the dimensions of uncertainty will contribute to the field of clinical genomics by facilitating recognition of the uncertainties inherent in each step of genomic testing and helping researchers, clinicians, patients, and relatives establish realistic expectations of its processes and outcomes.4 It will also guide future research by helping identify and close key knowledge gaps. INTRODUCTION The current study used as its starting point a conceptual taxonomy of uncertainty in health care.1 This taxonomy begins with a working definition of “uncertainty” as the conscious awareness of ignorance—a self-awareness of incomplete knowledge of some aspect of the world.5 It then distinguishes three major dimensions of uncertainty: I) source, II) issue, and III) locus. Source refers to the cause of a given uncertainty or the fundamental reason for a specific knowledge gap. Issue refers to the substantive situation, outcome, or alternative to which a given uncertainty applies. Locus is the particular party or stakeholder in whose mind(s) a given uncertainty resides.1 The taxonomy further divides each of these dimensions into more specific sub-dimensions by which various uncertainties can be categorized. The long-term goal of this multi-dimensional conceptual taxonomy is to facilitate a clearer understanding of the uncertainties inherent in the diagnosis and prognosis of various disorders, and a rational approach to their management by researchers, clinicians, patients, and other stakeholders.1,6 Author Manuscript Genet Med. Author manuscript; available in PMC 2017 August 07. Genet Med. Author manuscript; available in PMC 2017 August 07. MATERIALS AND METHODS Author Manuscript The current project was conceptual in nature, and aimed to develop a systematic and logically coherent working taxonomy of uncertainty in CNGS that can be refined and modified over time. It was empirically informed by a synthesis of insights from open-ended qualitative interviews undertaken with leaders in the clinical genomics field. These interviews were conducted by one of the investigators (PKJH) between June and August 2014 with six genomics, genetic counseling, and bioethics experts from among the NHGRI- funded Clinical Sequencing Exploratory Research (CSER) consortium investigators who volunteered to participate. The interviews explored experts’ views on the major sources and issues of uncertainty in clinical genomics, the reducibility or irreducibility of these uncertainties, and their implications for clinical care and the lives of patients and families. Interviews were carried out until saturation of novel topics was achieved. The interviews were transcribed verbatim and the transcripts coded for these topics. Themes were then synthesized from the coded transcripts by two of the study investigators (BBB, PKJH), and used to expand a draft taxonomy of uncertainties specific to genome sequencing and clinical genomic testing that was modeled on Han and colleagues’ three-dimensional conceptual taxonomy. Four of the expert interviewees also reviewed and helped to iteratively refine the taxonomy. Author Manuscript In parallel to our initial effort to represent the key uncertainties of clinical genomics within a conceptual taxonomy, we developed an interactive taxonomy platform website (http:// Han et al. Page 4 research.nhgri.nih.gov/taxonomy/). The purpose of this website is to disseminate the new uncertainty taxonomy and collaboratively engage researchers in its further refinement. The interactive web page is curated by three of the authors (BBB, LGB, PKJH), and enables individuals to suggest revisions. Using a platform that allows ongoing input enables the taxonomy to reflect the current experiences and knowledge of a broad range of individuals, and to be responsive to technological and conceptual advances. In a field as dynamic as clinical genomics, this capacity is essential and enhances the representativeness and utility of the taxonomy. Author Manuscript Dimension 1: Sources of uncertainty Author Manuscript Author Manuscript Clinical ambiguity, the final principal sub-type of ambiguity, arises from incomplete or erroneous family or clinical history data. These data limitations have multiple causes including missing information, faulty information gathering, or problems of recall. Complexity, the third main source of uncertainty in clinical genomic testing, has three principal sub-types reflecting distinct origins: 1) multiplicity of causes, 2) multiplicity of effects, and 3) effect modification. Multiplicity of causes, in turn, arises from three specific sub-sources. The first is locus heterogeneity: the phenomenon in which a single disorder or phenotypic characteristic is caused by mutations in heterogeneous genes in different individuals (e.g., autism). The second is complex genetic traits: the determination of single disorders or phenotypic characteristics by variation at multiple genetic loci (e.g., height). The third is non-genetic causation: the presence of non-genetic (i.e., environmental) determinants of disorders or phenotypic characteristics, which may interact with genetic determinants and have poorly quantified effects. Author Manuscript Multiplicity of effects, the second principal sub-type of complexity, originates from pleiotropy: a phenomenon in which a single gene mutation causes multiple apparently unrelated disorders or phenotypic characteristics (e.g., Neurofibromatosis). Effect modification, the final principal sub-type of complexity, reflects the presence of moderating or mediating causal pathways such as gene by environment interactions, which affect the extent to which genomic factors affect health. Importantly, any or all of these major sources of uncertainty—i.e., probability, ambiguity, complexity—and the more specific sub-types and problems from which uncertainty ultimately originates may apply, in principle, to any given CNGS test. However, these uncertainty sources do not manifest in the abstract, but in particular issues that constitute the second major dimension of uncertainty. Author Manuscript Genet Med. Author manuscript; available in PMC 2017 August 07. Dimension 1: Sources of uncertainty The first of the three major dimensions of uncertainty is source, “which is further subdivided into three main types: probability, ambiguity, and complexity (Figure 1). Probability (otherwise known as “risk”) refers to the fundamental indeterminacy or stochastic nature of future outcomes, and has also been termed “first-order” or “aleatory” uncertainty; an example is the point estimate of risk (e.g., “20% probability of benefit from treatment”). Ambiguity refers to the lack of reliability, credibility, or adequacy of information about probability and is also known as “second-order” or “epistemic” uncertainty. Ambiguity arises in situations in which risk information is unavailable, inadequate, or imprecise; a predominant representation is the confidence interval around a point estimate (e.g., “10% to 30% probability of benefit from treatment”). Complexity refers to features of risk information that make it difficult to understand; examples include conditional probabilities or multiplicity in risk factors, outcomes, or choice options, which diminish their comprehensibility or produce information overload.” Author Manuscript Because probability arises from the fundamental indeterminacy of future events, there is no additional source into which it can be further subdivided. However, the other main sources of uncertainty, ambiguity and complexity, can be further categorized according to more specific fundamental types and sources (Figure 1). Ambiguity in clinical genome sequencing has three principal sub-types: 1) conceptual, 2) methodological, and 3) clinical. Conceptual ambiguity arises from two main sources. The first is model inadequacy: limitations in either the theoretical or empirical models (e.g., genes, animal systems) used to represent gene- disease mechanisms. The second is nosological inadequacy: limitations in the conceptual systems used to classify diseases and phenotypes.4 Methodological ambiguity, the second principal sub-type of ambiguity, arises from five main sub-sources: 1) sample or data integrity problems, 2) test limitations, 3) unmeasured factors, 4) procedural variability or error, and 5) test misinterpretation. Sample or data integrity problems represent deficiencies in laboratory samples or processing, which result in diagnostic error. Test limitations represent inherent constraints in the accuracy or precision of laboratory instruments or techniques. Unmeasured factors are causal biological variables that affect phenotype, but are not currently recognized or assayed. Procedural variability or error refers to methodological problems that leave existing variants undetected. Test misinterpretation refers to failures of laboratory or clinical personnel to correctly annotate or Author Manuscript Han et al. Page 5 analyze genomic test results, or of health care providers to accurately understand the clinical implications of the results. Dimension 2: Issues of uncertainty The second of the three major dimensions of uncertainty is issue—the specific substantive matter about which an individual lacks knowledge. Issues of uncertainty can further be subdivided into three main types: scientific, practical, and personal (Figure 1). “Scientific uncertainty is data-centered, whereas practical and personal uncertainties are system- and patient-centered, respectively.”1 Author Manuscript In CNGS scientific uncertainty pertains to many different issues—diagnostic, prognostic, causal, or therapeutic. Diagnostic issues include: 1) Gene-phenotype association– identification of variants in a gene that cause disease and detection of a phenotype; 2) Pathogenicity of variants—whether or not a variant causes disease; and 3) Phenotype- disease association—whether a given phenotypic manifestation is part of a disease or syndrome. Prognostic issues include the future outcomes of individuals and their family members. Causal issues include the underlying factors and mechanisms that determine or explain a given genomic variant or its ultimate phenotypic manifestations. Therapeutic Page 6 Han et al. Page 6 issues include the prevention and treatment of the diseases or health consequences of genomic variants. issues include the prevention and treatment of the diseases or health consequences of genomic variants. Author Manuscript Author Author Manuscript Personal uncertainty pertains to psychosocial and existential issues including the effects and implications of genomic test results on one’s own goals or outlook on life, personal relationships, sense of meaning, or one’s future well-being and that of one’s family members or social groups. An over-simplified understanding of results by patients and their relatives would also be reflected in this domain. Personal uncertainties may also encompass moral questions—e.g., whether the results of CNGS testing ought to be disclosed to particular individuals or shared with family members or other parties. Practical uncertainty pertains to lack of knowledge about both the structures of health care— i.e., the institutional facilities and resources of the health care system—and the processes of health care—i.e., the procedures and actions required to deliver, access, or utilize health care services related to CNGS testing. Author Manuscript Importantly, for all these specific issues of uncertainty—scientific, practical, and personal— the underlying cause may be any of the sources comprising the first dimension of uncertainty (probability, ambiguity, complexity). Dimension 2: Issues of uncertainty Any or all of these sources may engender uncertainty about not only diagnosis, prognosis, causal explanations, and treatment recommendations (scientific uncertainty), but also the expected quality of care and the procedures required to access care (practical uncertainty), as well as the effects of illness or treatment on one’s personal relationships and goals in life (personal uncertainty). In theory, probabilities exist for all of these outcomes, although these probabilities are unknown to varying degrees—and thus ambiguous—and further compounded by varying degrees of complexity. Author Manuscript Genet Med. Author manuscript; available in PMC 2017 August 07. A case example: Varieties of uncertainty in clinical genomic testing To illustrate the potential value of our provisional conceptual taxonomy of uncertainty, we present a case (Box 1). It demonstrates the variety of uncertainties that arise in CNGS testing, and how the taxonomy may provide a useful organizational framework that can make existing uncertainties more explicit to different stakeholders. This case involves numerous areas of uncertainty that are differentially perceived—resulting in varying degrees of uncertainty—by individual stakeholders, each of whom represents a distinct locus of uncertainty. We now use our taxonomy to compare and contrast the uncertainties experienced by three of the different stakeholders: clinicians, patients, and genetics laboratory professionals. Author Manuscript Clinician perspective—Mark’s internist’s primary concern is the care of her patient. She wonders about what clinical actions are appropriate, and how to counsel Mark at this point. She recognizes that multiple uncertainties apply to Mark’s case, but her own greatest uncertainties revolve around the strength of the evidence about the risk of myopathy associated with Mark’s particular genomic variant, and thus the appropriateness of recommending a lifestyle change and/or medication for Mark (Box 2). The most salient sources and issues of uncertainty for this internist fall in several places in our conceptual taxonomy, and influence the content of the counseling she provides to the patient. She focuses her discussion on the evidence for the risk of drug-induced myopathy and the known approaches to mitigating this risk. Patient perspective—Mark’s primary concern focuses on the implications of his genomic variant and the actions he needs to take to preserve his health. Note that many of the uncertainties that Mark experiences (Box 3) are identical to those that his internist faces. However, because of Mark’s relative scientific naïveté, the sources of these uncertainties are less differentiated or precise. At the same time, some of Mark’s uncertainties—notably his personal uncertainties—initially reside in only his mind and not the internist’s, because it is Mark whose own life is affected, and he is the expert on his own values and perspectives. Genetics laboratory perspective—In contrast to clinicians and patients, laboratory professionals are primarily concerned with technical and scientific aspects of CNGS, and its implications that extend beyond the care of any individual patient. The laboratory professional’s perspective on uncertainty is focused on the bioinformatics, the testing pipeline, and the classification of variants (Box 4). Dimension 3: Loci of uncertainty The third dimension of the taxonomy, the locus of uncertainty—i.e., the party in whose mind uncertainty resides—can be any of a number of different stakeholders: patients, clinicians, researchers, or other individuals including family members, regulators, payers, or health policymakers (Figure 1). Uncertainty may be shared, as when a physician effectively educates a patient about limitations in scientific evidence regarding the benefits, harms, and clinical utility of CNGS testing. At other times, however, uncertainty may not be shared between parties, as when physicians (but not their patients) are aware of scientific ignorance, or when patients (but not their physicians) are aware of ignorance about their own values and preferences. Author Manuscript The deeper challenge, however, is that the differential salience of specific uncertainties to individual stakeholders can perpetuate ignorance. When individuals ignore their uncertainty about a given issue, they diminish their awareness of what they do not know. This can be consequential, particularly in clinical and policy endeavors—where different domains of knowledge (and ignorance) intersect. In the domain of clinical care, for example, patients who do not grapple with scientific uncertainties surrounding the benefits and harms of medical interventions cannot benefit fully from shared decision making. Han et al. Page 7 A comprehensive taxonomy of uncertainty in clinical genomic testing can mitigate this problem by making different stakeholders more aware of—and better able to understand— areas of uncertainty that lie beyond their usual focus of attention. The sharing of uncertainties that such a taxonomy can facilitate may, in turn, help people make better informed decisions about how to manage these uncertainties. A comprehensive taxonomy of uncertainty in clinical genomic testing can mitigate this problem by making different stakeholders more aware of—and better able to understand— areas of uncertainty that lie beyond their usual focus of attention. The sharing of uncertainties that such a taxonomy can facilitate may, in turn, help people make better informed decisions about how to manage these uncertainties. Author Manuscript Genet Med. Author manuscript; available in PMC 2017 August 07. Bridging perspectives on uncertainty in CNGS The foregoing case illustrates how the various stakeholders who represent the loci of uncertainty vary in the breadth, depth, and precision of their awareness of areas for which there is a lack of knowledge. This variation in uncertainty reflects differences in the prior knowledge each stakeholder brings to CNGS and in their primary concerns, both professional and personal. These differences can leave stakeholders unaware of important areas of ignorance, and thus to have false certainty. For example, Mark may be uninformed about scientific ambiguity regarding the strength of the association between his variant and his risk for adverse health outcomes. He may assume that if information about his variant was returned to him with the recommendation that he avoid certain statin therapy, then there is relative certainty about the risk for myopathy. For him, the consequences of medication therapy may be most salient. At the same time, Mark’s internist may be uninformed about outcomes important to Mark, such as the personal, social, and economic consequences of interventions undertaken to reduce Mark’s disease risks. Finally, genetics laboratory professionals may be uninformed about the concerns, values, and needs of individual patients. Author Manuscript Our conceptual taxonomy can help mitigate these problems by bridging the perspectives of different stakeholders—thereby promoting shared awareness of otherwise unconsidered sources and issues of uncertainty, and enabling stakeholders to approach these uncertainties in an organized manner. For example, the taxonomy may facilitate clinicians’ awareness of the various uncertainties identified by genetics laboratories, in order to better differentiate areas of greater or lesser certainty and to weigh the evidence more explicitly and precisely in making clinical recommendations for patients. This exercise may also prompt the internist to return to the primary literature to update her knowledge on the areas of uncertainty about penetrance. It can also help genetics professionals identify important knowledge gaps to frame their next study related to use of CNGS, or efforts to improve procedures for implementing CNGS. The taxonomy can also help clinicians organize their approach to counseling patients and promoting shared decision making. It supplies a framework for the discussion and a reminder of important and salient issues that the discussion should address to maximize the patient’s ability to share in the making of an informed decision about further action. A case example: Varieties of uncertainty in clinical genomic testing Laboratory geneticists have access to greater knowledge about all of the methodological issues that produce uncertainty in CNGS, and their broader concerns also make other uncertainties more salient. For these reasons it is also deeper scientifically, encompassing more specific questions and precisely defined sources of ignorance. Yet it is also more generic—i.e., focused on generalizable knowledge Author Manuscript Page 8 Page 8 Han et al. affecting populations more than specific knowledge affecting individuals. Whereas the uncertainties experienced by Mark and his internist focus on the implications of CNGS for Mark himself, the uncertainties of laboratory professionals, researchers, and policymakers extend beyond Mark or any given individual. Author Manuscript Genet Med. Author manuscript; available in PMC 2017 August 07. Bridging perspectives on uncertainty in CNGS At the same time, the taxonomy can draw clinicians’ attention to potential sources or issues of uncertainty that may not affect clinicians themselves, but may have profound effects on patients. For example, Mark’s internist may not feel at all uncertain about her recommendations to mitigate risks of the identified variant; however, questions may remain in Mark’s mind. Similarly, the internist may not experience uncertainty about personal issues that are important to Mark—e.g., doubts about whether having his variant will affect his lifestyle, or about whether he should share this information with his wife who tends to worry Author Manuscript Page 9 Han et al. Page 9 about his health. The internist can use the taxonomy as a mental checklist that reduces the potential for her to overlook uncertainties that exist in her patient’s mind and that should be explored further to promote shared decision making. The taxonomy can help clinicians and patients reach greater mutual understanding of not only which uncertainties exist in each other’s minds, but also which uncertainties each party judges to be important. Author Manuscript Author Author Manuscript At the same time, the taxonomy can also facilitate a broader understanding of CNGS-related uncertainties among other stakeholders, whose unique concerns and perspectives might limit their awareness of existing sources or issues of uncertainty. For example, laboratory professionals may naturally focus more on ambiguity arising from technical limitations in genomic sequencing tests and error rates in the procurement and processing of biological specimens. Researchers, on the other hand, may focus on ambiguity arising from conceptual problems (e.g., the distinctions between normal and abnormal phenotypes, non-pathogenic and pathogenic genotypes) or methodological problems (e.g., how sequencing is conducted, how sequencing data are analyzed and interpreted). Payers may be most concerned about whether recommended testing meets their internal criteria for reimbursement. Health policymakers may focus primarily on ethical uncertainties (e.g., whether and when sequencing data should be disclosed to patients and family members, and used as a basis for medical intervention), as well as the societal consequences of implementing genomic sequencing. The primary value of the new taxonomy is to enable all of these different stakeholders to better acknowledge—and thereby understand—the uncertainties that apply to not only their own domains of interest, but also those of other stakeholders. Author Manuscript A critical question, however, is how broad and deep the awareness of ignorance ought to be for any given stakeholder. Bridging perspectives on uncertainty in CNGS Human attention spans are limited, and uncertainty can have adverse psychological effects on individuals causing fear, diminished sense of well-being, and avoidance of decision making.1,6 The appropriate breadth and depth of uncertainty to convey to different stakeholders remains to be determined, and likely depends on their unique goals and concerns. Author Manuscript Genet Med. Author manuscript; available in PMC 2017 August 07. DISCUSSION We have proposed a new taxonomy of medical uncertainty in genome sequencing as a tool to help standardize how uncertainty is conceptualized, defined, and described by various stakeholders motivated by differing goals, perspectives, and concerns. Building upon an existing taxonomy of medical uncertainty,1 the current effort aims to help diverse stakeholders undertake a rational, logically coherent approach to understanding and managing uncertainty in CNGS. The new taxonomy can enable stakeholders to identify and assess uncertainties that exist but may escape their awareness, and to thereby determine the best course of action—e.g., information seeking and further scientific research for uncertainties that are reducible, and strategies to promote coping and adaptation for uncertainties that are irreducible. Author Manuscript Indeed, we believe that a taxonomic approach to uncertainty may benefit other domains of medicine, and the novel nature of CNGS provides an opportunity to model such an approach and demonstrate its broader utility. Applying a conceptual taxonomy of uncertainty to Han et al. Page 10 CNGS may highlight questions and concerns related to the novel nature of this technology, and applying the taxonomy to other novel technologies might have similar effects. The taxonomy will also require continual revision as the state of the science in CNGS advances, diminishing some uncertainties.7 Meanwhile, for patients and research participants deciding whether to undergo CNGS, uncertainties about its benefits and risks need to be explicitly acknowledged and communicated to ensure informed decision making—a requirement both in CNGS and in medicine more broadly. Applying our conceptual taxonomy in consent discussions between genetic counselors or research nurses, and patients may enhance informed decision making by increasing the precision of patients’ understanding of the uncertainties in genome sequencing—e.g., contrasting reducible and non-reducible uncertainties of different types and significance. Furthermore, in the disclosure of CGNS results, the taxonomy can help clinicians and patients understand what uncertainties need to be accepted and managed clinically and psychologically, and facilitate actions to accomplish these goals. Author Manuscript Aut Author Manuscript Identifying the dimensions of uncertainty in a consistent manner applicable to all kinds of medical interventions, genomic and non-genomic, can help various stakeholders have a clearer and more realistic understanding of the value and limitations of both CNGS and other medical interventions, and make better informed decisions about their use. Our taxonomy of uncertainty in CNGS is an initial step toward this broader goal. Acknowledgments The authors thank Dave Kanney and Tyra Wolfsberg for designing the web-based platform for the taxonomy, Sharon Plon for her interview and suggestions for the manuscript, and Barbara Athens for early diagrams of the taxonomy. The Intramural Research Program at the National Human Genome Research Institute, National Institutes of Health funded this study. Dr. Green is supported by NIH grants: U01HD006500, U19HD077671, R01HG005092, R01HG02213, U01HG008685 and R01-HG006615, R01CA154517 and U41HG006834. Author Manuscript DISCUSSION Furthermore, because medical knowledge is continuously evolving and limited by the perspectives of different stakeholders, any taxonomy of uncertainty is at best provisional, incomplete, and subject to revision. Accordingly, we have launched a website that provides stakeholders with the opportunity to edit and refine the taxonomy to enhance its usefulness to the broader community. Comments will be stored, curated, and used to periodically update the taxonomy. It will thus be a dynamic and consensual effort undertaken by many different stakeholders. Author Manuscript The novelty of CNGS and the vast amount of genetic code yet to be deciphered makes the uncertainty surrounding this technology inevitable. Our current conceptual taxonomy draws attention to this uncertainty; however, it is offered not to discourage use of CNGS, but to provide a framework that can inform efforts to reduce uncertainties and ultimately promote its confident and appropriate use. It remains for future work to refine and apply the taxonomy in efforts to study and utilize CNGS to improve health. Genet Med. Author manuscript; available in PMC 2017 August 07. 1. Han PKJ, Klein WMP, Arora NK. Varieties of uncertainty in health care: a conceptual taxonomy. Med Decis Making. 2011; 31:828–838. [PubMed: 22067431] Genet Med. Author manuscript; available in PMC 2017 August 07. 7. Vassy JL, Korf BR, Green RC. How to know when physicians are ready for genomic medicine. Sci Transl Med. 2015; 7:287fs19. References Genet Med. Author manuscript; available in PMC 2017 August 07. Han et al. Page 11 Page 11 2. de Ligt J, Willemsen MH, van Bon BWM, et al. Diagnostic exome sequencing in persons with severe intellectual disability. N Engl J Med. 2012; 367:1921–1929. [PubMed: 23033978] Author Manuscript 3. Biesecker LG, Green RC. Diagnostic clinical genome and exome sequencing. N Engl J Med. 2014; 370:2418–2425. [PubMed: 24941179] 4. Bowker, GC., Star, SL. Sorting things out: classification and its consequences. Cambridge: MIT Press; 1999. 5. Smithson, M. Ignorance and uncertainty: emerging paradigms. Berlin: Springer Verlag; 1989. 6. Han PKJ. Conceptual, methodological, and ethical problems in communicating uncertainty in clinical evidence. Med Care Res Rev. 2013; 70:14S–36S. [PubMed: 23132891] 6. Han PKJ. Conceptual, methodological, and ethical problems in communicating uncertainty in clinical evidence. Med Care Res Rev. 2013; 70:14S–36S. [PubMed: 23132891] 7. Vassy JL, Korf BR, Green RC. How to know when physicians are ready for genomic medicine. Sci Transl Med. 2015; 7:287fs19. 7. Vassy JL, Korf BR, Green RC. How to know when physicians are ready for genomic medicine. Sci Transl Med. 2015; 7:287fs19. Author Manuscript Author Manuscript Author Manuscript Han et al. Page 12 Box 1 Pharmacogenetics case of statin-induced myopathy risk A 37-year-old man, Mark, has moderate, untreated hypercholesterolemia that is being managed by diet with some degree of success, but he has not been able to achieve his target cholesterol levels. Mark’s internist ordered CNGS because Mark’s son has autism. Mark’s sequence was accessed primarily for filtering variants to exclude candidates for the cause of his son’s autism. However, Mark’s internist requests an extended genome analysis that identifies Mark as having a chr12:21,331549T>C, SLCO1B1 p.(Val174Ala) heterozygous variant, which predicts an increased risk for myopathy from statin (especially simvastatin). After learning about the possible side effect of myopathy, Mark is concerned that this treatment approach could compromise his running ability and interfere with his regular social contact with running companions. Note: The authors devised this hypothetical case to illustrate application of the taxonomy. It was not based on an actual person, and any potential resemblance to an actual case is coincidental. Box 1 Author Manuscript Author Manuscript Genet Med. Author manuscript; available in PMC 2017 August 07. Genet Med. Author manuscript; available in PMC 2017 August 07. Pharmacogenetics case of statin-induced myopathy risk A 37-year-old man, Mark, has moderate, untreated hypercholesterolemia that is being managed by diet with some degree of success, but he has not been able to achieve his target cholesterol levels. Mark’s internist ordered CNGS because Mark’s son has autism. Mark’s sequence was accessed primarily for filtering variants to exclude candidates for the cause of his son’s autism. However, Mark’s internist requests an extended genome analysis that identifies Mark as having a chr12:21,331549T>C, SLCO1B1 p.(Val174Ala) heterozygous variant, which predicts an increased risk for myopathy from statin (especially simvastatin). After learning about the possible side effect of myopathy, Mark is concerned that this treatment approach could compromise his running ability and interfere with his regular social contact with running companions. Author Manuscript Note: The authors devised this hypothetical case to illustrate application of the taxonomy. It was not based on an actual person, and any potential resemblance to an actual case is coincidental. Author Manuscript Author Manuscript Author Manuscript Han et al. Page 13 Box 2 Clinician perspective I. Source A. Probability: Mark’s genomic variant confers some risk of drug- induced myopathy. B. Ambiguity: The strength of the scientific evidence linking Mark’s genomic variant with drug-induced myopathy is not clear. 2 Methodological c Unmeasured factors: Associations with additional biological variables that may play a causal role have not been established. C. Complexity 3 Effect modification a. Gene by environment interactions: Mark’s exercise program and other environmental and behavioral factors may moderate the risk of drug- induced myopathy. II. Issue A. Scientific 1 Diagnostic a Phenotype-disease association: Whether Mark’s hypercholesterolemia will result in cardiovascular disease; whether Mark’s genomic variant will result in drug-induced myopathy. 3 Prognostic b Individual: Whether Mark will develop myopathy and if so, how severe it will be. 4 Therapeutic a. Prevention: What interventions can be undertaken to avert drug-induced myopathy or cardiovascular disease. b. Treatment: Whether statin therapy should be prescribed and if so, which statin and what dose. Author Manuscript Author M Author Manuscript Clinician perspective I. Source A. Probability: Mark’s genomic variant confers some risk of drug- induced myopathy. B. Ambiguity: The strength of the scientific evidence linking Mark’s genomic variant with drug-induced myopathy is not clear. 2 Methodological c Unmeasured factors: Associations with additional biological variables that may play a causal role have not been established. C. Complexity 3 Effect modification a. Genet Med. Author manuscript; available in PMC 2017 August 07. Pharmacogenetics case of statin-induced myopathy risk Gene by environment interactions: Mark’s exercise program and other environmental and behavioral factors may moderate the risk of drug- induced myopathy. II. Issue A. Scientific 1 Diagnostic a Phenotype-disease association: Whether Mark’s hypercholesterolemia will result in cardiovascular disease; whether Mark’s genomic variant will result in drug-induced myopathy. 3 Prognostic b Individual: Whether Mark will develop myopathy and if so, how severe it will be. 4 Therapeutic a. Prevention: What interventions can be undertaken to avert drug-induced myopathy or cardiovascular disease. b. Treatment: Whether statin therapy should be prescribed and if so, which statin and what dose. II. Issue Author Manuscript Author Manuscript Han et al. Page 14 Box 3 Patient perspective I. Source A Probability: Mark’s genomic variant confers some risk of drug- induced myopathy. C Complexity: Information about Mark’s genomic variant is unfamiliar, multi-faceted, and thus difficult for him to understand. II. Issue A. Scientific 1 Diagnostic: The meaning of Mark’s genomic variant is unclear to him. 3 Prognostic b Individual: Whether Mark will develop myopathy and if so, how severe it will be. 4 Therapeutic a. Prevention: What interventions can be undertaken to avert drug-induced myopathy or cardiovascular disease. b. Treatment: Whether statin therapy should be prescribed and if so, which statin and what dose. B. Personal 1. Psychological: Whether and to what extent Mark will experience adverse effects of knowledge of the genomic variant on his psychological well-being. 2. Social: Whether discontinuing running with Mark’s social group will have adverse effects on his social well-being. C. Practical: What actions Mark needs to undertake, both now and in the future, to access the care he needs is unclear. Author Manuscript Author Author Manuscript Patient perspective Author Manuscript 1. Psychological: Whether and to what extent Mark will experience adverse effects of knowledge of the genomic variant on his psychological well-being. C. Practical: What actions Mark needs to undertake, both now and in the future, to access the care he needs is unclear. Author Manuscript Han et al. Page 15 Box 4 Laboratory scientist perspective I. Source B Ambiguity 1. Conceptual a Model inadequacy: The myopathy risk model does not account for all causal pathways and mechanisms. 2. Methodological c Unmeasured factors: Other pharmacogenomic variants that may increase or decrease the likelihood of drug-induced myopathy (e.g., ABCB1 efflux transporter) are not detected by the assay. Genet Med. Author manuscript; available in PMC 2017 August 07. Genet Med. Author manuscript; available in PMC 2017 August 07. Pharmacogenetics case of statin-induced myopathy risk C Complexity 1 Multiplicity of Causes b Complex genetic trait: Unrecognized variants may affect the risk of drug-induced myopathy. 3 Effect modification a Gene by environment interactions: Environmental and behavioral factors may moderate the risk of drug-induced myopathy. II. Issue A Scientific 1. Diagnostic b Pathogenicity of variants: Whether a given genomic variant will result in drug-induced myopathy. 2. Causal: How and why statins cause myopathy. 3. Prognostic b Individual: Whether any specific individual characteristics are significant predictors of myopathy onset and severity. C Practical 1. Procedural Author Manuscript Author M Author Manuscript Author Manuscript Author Manuscript Genet Med. Author manuscript; available in PMC 2017 August 07. Genet Med. Author manuscript; available in PMC 2017 August 07. a. Genomic testing: The extent to which testing facilities adhere to standards for the conduct and interpretation of CNGS. Han et al. Page 16 a. Genomic testing: The extent to which testing facilities adhere to standards for the conduct and interpretation of CNGS. Han et al. Page 16 Han et al. Page 16 a. Genomic testing: The extent to which testing facilities adhere to standards for the conduct and interpretation of CNGS. Author Manuscript Author Manuscript Author Manuscript Han et al. Page 17 Figure 1. A Taxonomy of Uncertainty This is a visual representation of a three-dimensional taxonomy of medical uncertain clinical genome sequencing. The three major dimensions are source, issue, and locus Author Manuscript Author Manuscript Author Manuscript Author Manuscript Figure 1. A Taxonomy of Uncertainty Author Manuscript g y y This is a visual representation of a three-dimensional taxonomy of medical uncertainty in clinical genome sequencing. The three major dimensions are source, issue, and locus.
https://openalex.org/W4381279714	https://www.researchsquare.com/article/rs-2534270/latest.pdf	English	null	A resilient workforce: patient safety and the workforce response to a cyber-attack on the ICT systems of the national health service in Ireland	Research Square (Research Square)	2,023	cc-by	6,759	A resilient workforce: patient safety and the workforce response to a cyber-attack on the ICT systems of the national health service in Ireland Gemma Moore National Quality and Patient Safety Directorate, Health Service Executive Zuneera Khurshid UCD IRIS Centre, School of Nursing, Midwifery and Health Systems, University College Dublin Therese McDonnell (  therese.mcdonnell@ucd.ie ) UCD IRIS Centre, School of Nursing, Midwifery and Health Systems, University College Dublin Lisa Rogers UCD IRIS Centre, School of Nursing, Midwifery and Health Systems, University College Dublin Orla Healy National Quality and Patient Safety Directorate, Health Service Executive UCD IRIS Centre, School of Nursing, Midwifery and Health Systems, University College Dublin Therese McDonnell (  therese.mcdonnell@ucd.ie ) UCD IRIS Centre, School of Nursing, Midwifery and Health Systems, University College Dublin Orla Healy Results The impact of the cyber-attack varied across services depending on the type of care being offered, the reliance on software, and local IT support. Staff stepped up to the challenges and quickly developed and implemented innovative solutions, exhibiting great resilience, teamwork, and adaptability with a sharp focus on ensuring patient safety. The cyber-attack resulted in a flattening of the healthcare hierarchy, with shared decision-making at local levels leading to an empowered frontline workforce. However, participants in this study felt the stress placed on staff by the attack was more severe than the cumulative effect of the COVID-19 pandemic. Research Article Posted Date: May 30th, 2023 DOI: https://doi.org/10.21203/rs.3.rs-2534270/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Additional Declarations: Competing interest reported. OH and GM are employees of the Health Service Executive. OH is the National Clinical Director of the Directorate and GM is responsible for Qualitative Research and Evaluation. The remaining authors, TMD, LR and ZK, declare no competing interests. Version of Record: A version of this preprint was published at BMC Health Services Research on October 17th, 2023. See the published version at https://doi.org/10.1186/s12913-023-10076-8. Page 1/16 Conclusions Limited contingencies within the health system IT infrastructure - what we call a lack of system resilience - was compensated for by a resilient workforce. Within the context of the prevailing COVID-19 pandemic, this was an enormous burden on a dedicated workforce. The adverse impact of this attack may have long-term and far-reaching consequences for staff wellbeing. Design and investment in a resilient health system must be prioritised. Background In May 2021, the Irish public health service was the target of a cyber-attack. The response by the health service resulted in the widespread removal of access to ICT systems. While services including radiology, diagnostics, maternity, and oncology were prioritised for reinstatement, recovery efforts continued for over four months. This study describes the response of health service staff to the loss of ICT systems, and the risk mitigation measures introduced to safely continue health services. The resilience displayed by front line staff whose rapid and innovative response ensured continuity of safe patient care is explored. Methods To gain an in-depth understanding of staff experiences of the cyber-attack, eight focus groups (n = 36) were conducted. Participants from a diverse range of health services were recruited, including staff from radiology, pathology/labs, radiotherapy, maternity, primary care dental services, health and wellbeing, COVID testing, older person’s care, and disability services. Thematic Analysis was applied to the data to identify key themes. Introduction Health care professionals are the resource that underpin the delivery of safe and high-quality care in a resilient health system. However, resilient healthcare does not focus on the individual’s capacity to cope but rather on what enables the workers, team, and unit or organisation to adapt and cope effectively in Page 2/16 different situations (1). While definitions and concepts of health system resilience differ substantially throughout the literature, all have a common foundation: they regard resilience as the degree of change a system can undergo while maintaining its functionality (2, 3). Health system resilience is not a single dimension but rather an emergent property of the health system as a whole (4), and requires a strong and committed health workforce, characterised by healthcare personnel who show up for work that might be difficult and, in certain contexts, dangerous (4). Health system resilience has also been defined as the capacity to absorb, adapt, and transform when exposed to a shock, such as a pandemic, natural disaster or armed conflict and still retain the same control over its structure and functions (5). While continuing to respond to the challenges presented by the COVID-19 pandemic, the Irish public health system experienced a significant further shock to the delivery of healthcare, when access to ICT systems across most services nationally ceased. On Friday, 14th May 2021, the Health Service Executive (HSE) of Ireland was the target of a cyber-attack, a criminal infiltration of the HSE’s IT systems using Conti ransomware. In response, the HSE invoked its Critical Incident Process, leading to the decision to switch off all HSE ICT systems and disconnect the National Healthcare Network (“NHN”) from the internet (6). The HSE is responsible for the national public health service in Ireland, serving a population of 5.1 million people. As the largest employer in the country, the HSE employs over 130,000 staff directly and indirectly, with over 70,000 devices such as laptops and PCs in use (6). The public health service is provided at approximately 4,000 locations, including 54 acute hospitals. Invoking the Critical Incident Process immediately resulted in healthcare professionals losing access to all HSE provided IT systems - including patient information, clinical care and laboratory systems. Non-clinical systems such as financial systems, payroll and procurement systems were also lost. Normal communication channels such as email and networked phone lines were no longer available (6). Methods The HSE National Quality & Patient Safety Directorate commissioned a mixed-methods research study to understand the clinical impact of the Conti cyber-attack on patient safety (8). This paper presents the findings of one aspect of this overall study, a qualitative analysis of focus groups exploring the experiences of staff working in acute, maternity, and community settings. Recruitment Different settings included in the study were a large university hospital, a maternity teaching hospital, and various sites from one Community Healthcare Organisation (CHO). In consultation with the relevant Heads of Service for Quality Safety and Service Improvement, representatives from services in these sites most affected by the cyber-attack were invited to participate in focus groups to share their experiences and learning. Purposeful sampling identified clinicians, executive managers, scientists and other administrative and support staff who were invited to participate voluntarily. This included radiology, pathology/labs, radiotherapy, maternity, primary care dental services, health and wellbeing, COVID testing, older person’s care, and disability services. A detailed study information sheet was provided (Appendix File 1) to potential participants and staff self-selected to participate. Sample size was determined in consultation with Quality and Safety, hospital and community service managers. Data saturation was identified early during analysis which confirmed the adequacy of the sample size. Introduction Therefore, this attack and the sudden removal of ICT system access presented a monumental challenge to staff tasked with continuing to deliver safe healthcare. The impact of this attack on the Irish health system is illustrated by a framework proposed by Thomas et al (2020) which sets resilience within the context of a 4-stage shock: preparedness; shock onset and acting rapidly; managing impact to preserve health system access and quality; and recovery and learning (7). While the HSE was not prepared for this cyber-attack (6), it did respond rapidly to limit the impact of the attack on IT systems nationally. Integrated governance structures were quickly established to oversee and expedite the clinical and operational response. Health services including patient administration systems, radiology, diagnostics, maternity, and oncology were prioritised to advance the resumption of systems (8). However, recovery efforts continued for over four months, a timescale far greater that initially expected (6), leaving many staff to manage the impact and preserve health system access and quality over a sustained period. The risk of cyber-attacks on health systems has amplified during the COVID-19 pandemic due to increased remote work and reliance on virtual methods of care delivery (9, 10). This study describes the response of staff and their teams within the health service to the loss of ICT systems due to the cyber- attack in May 2021, and the risk mitigation measures and contingencies introduced to safely continue Page 3/16 health and social care services. The resilience of healthcare staff who quickly adapted and innovated to ensure continuity of service and patient safety is explored. health and social care services. The resilience of healthcare staff who quickly adapted and innovated to ensure continuity of service and patient safety is explored. Results The inductive analysis of the eight focus groups, comprised of representatives from a broad range of health services (Table 1), generated six key themes within two broader categories: (1) continuity of patient care and (2) immediate and long-term consequences requiring action (Fig. 1). Continuity of Patient Care Healthcare staff worked diligently to ensure the continuation of services, at least at a basic level, while ICT systems remained inaccessible. Three themes were identified that explain the extent of the disruption to services and how the nature of the response by staff supported the resilience of the health system. Data Collection & Analysis Using an in-depth semi-structured topic guide (Appendix File 2), eight focus groups with 36 participants were conducted over a four-week period in September and October 2021, with each focus group lasting approximately 60 minutes. Focus groups were conducted by two experienced qualitative researchers virtually using online platforms (MS Teams and Cisco WebEx), depending on the preference of the participants due to Covid restrictions. Informed consent was sought, and participants were given the opportunity to ask questions and voice any concerns and asked to sign a consent form prior to the start of the focus groups (Appendix Files 3). The eight focus groups were conducted with staff working in clinical, business management, scientists, administrators, and information systems roles (Table 1). Page 4/16 Table 1 Focus group details Table 1 Focus group details Care Area Focus group Service type Number of participants Community Focus group 1 Dental Services 2 Focus group 2 Health and Wellbeing and COVID testing 4 Focus group 3 Disability 3 Focus group 4 Social Care – Older Persons 2 Acute Focus group 5 Radiotherapy 8 Focus group 6 Radiology 3 Focus group 7 Laboratory Services 4 Maternity Focus group 8 Maternity services 10 Total participants 36 Table 1 Focus group details Number of participants Thematic Analysis, as outlined by Braun and Clarke (11), guided the analysis structure. An inductive approach to coding was chosen to ensure the themes generated strongly reflected the data collected. TMD analysed the complete dataset, while LR double coded a random subset of the transcripts (n = 3). NVivo 12 software supported the analysis process. Variation in service disruption The impact of the cyber-attack varied across services depending on the reliance on software, the type of care being offered, and local IT support. The cyber-attack’s duration was unprecedented and backup systems contained basic patient data for the last 7–15 days, which quickly became irrelevant. Participants in acute and maternity services were completely reliant on electronic methods and were therefore more severely impacted: Page 5/16 Page 5/16 “our emails and other files and everything was gone. But they were the least of our worries now to be honest. So our lab information system was gone.” (Participant A12) “our emails and other files and everything was gone. But they were the least of our worries now to be honest. So our lab information system was gone.” (Participant A12) Patient information systems were not accessible, leaving clinical staff to treat patients without knowing their full history. Clinical care and laboratory systems could not be accessed, with clinicians “in the flick of a switch” (Participant A12) unable to carry out routine diagnostic procedures. There was a significant impact on radiotherapy services, with cessation of radiation treatment: “Most critically the patients who were on treatment having radiotherapy there was absolutely no way of continuing their treatment ..before we even got to that point .. there was no way of even identifying who they were to contact them not to come to their treatment.” (Participant A3) However, the effect of the cyber-attack was less severe for those working outside the acute services. In particular, the impact was “negligible” (Participant C7) for services still reliant on paper-based records due to low levels of computerisation. One unexpected outcome from the community care focus groups was that in the absence of ICT systems, staff found more time to spend with service users in residential homes: “you actually had people who could sit and talk to residents” (Participant C9). Staff commitment facilitating service continuity Staff commitment facilitating service continuity As the cyber-attack impacted their normal systems and practices, staff had to adapt to the challenge and implement and refine mitigations rapidly to ensure continuity of services: “So I suppose we mitigated as best we could with the resources we had to try and keep the patients as safe as possible, and that involved really lateral thinking, like thinking outside the box.” (Participant A7) Staff held regular, often daily, in-person meetings, which previously had been limited due to the prevailing pandemic. Staff took the initiative “to react and respond” (Participant C10) and made decisions locally. Solutions involved developing manual paper-based systems and forms, which required additional training by more experienced healthcare professionals as many staff members had no experience of recording on paper, having always worked within a computerised environment. Non-digital solutions included returning to outdated methods, with radiologists using “printing film” (Participant A11) and “carbon copy” (Participant A10). Common mitigations across all services included setting up Gmail accounts in the absence of HSE email, using personal phones and WhatsApp, conference calls, and using the postal service. The lack of patient information systems meant staff were not aware of who was due into the service and patients could not be individually contacted before attending the hospital. Therefore, notices were placed on external communication/websites requesting patients to phone in advance of scheduled appointments. Staff also risk-assessed patients and some services were postponed: “So what we did was we tried to stratify patients in terms of risk and anyone who was deemed low risk and an urgent appointment wasn't required, their outpatient appointments were then postponed until such Page 6/16 time as we had all the relevant details.” (Participant M3) Clinicians were forced to reconstruct very high dose radiotherapy relying on their recall, “we literally had only our memories to rely on” (Participant A6), to ensure the continuation of these critical treatments. Private hospitals were engaged to provide treatments, in particular to allow oncology patients to continue time-critical treatment. As priority systems began to return, staff worked tirelessly to ensure patients received much needed treatments: Clinicians were forced to reconstruct very high dose radiotherapy relying on their recall, “we literally had only our memories to rely on” (Participant A6), to ensure the continuation of these critical treatments. Private hospitals were engaged to provide treatments, in particular to allow oncology patients to continue time-critical treatment. Staff commitment facilitating service continuity As priority systems began to return, staff worked tirelessly to ensure patients received much needed treatments: “When we did get the service up and running…we’re normally a Monday to Friday service, we treated every weekend for a number of weekends to try and compensate for the gaps…anyone who had a gap in their treatment, they got treated every weekend for their remaining treatment”. (Participant A3) Immediate and long-term consequences requiring action Across all focus groups, healthcare staff raised concerns about the short and long-term consequences of the cyber-attack. Three themes were identified which reflect the impact of the response to the cyber-attack on employee wellbeing, shortcomings in the IT infrastructure and support, staff concerns about regaining productivity and the longer-term consequences of decisions made and manual processes implemented in the aftermath of the attack. Negative impact on healthcare staff The dedication of staff in response to the cyber-attack enabled the continuity of patient care in extremely difficult circumstances. Health service staff had just come through four waves of COVID-19 when the cyber-attack occurred, with participants reporting that staff continued to experience high levels of stress, anxiety, and uncertainty as they dealt with concurrent challenges of the COVID-19 pandemic and the cyber-attack. Clinical decision-making without the support of systems and data left many clinicians working very long hours and deeply concerned about the choices they we forced to make: “We were putting in 16 plus hour days, significantly more I think at the beginning, and then the uncertainties that impacted that. So, I think that is all I have to say about that. I will say I still wake up at 4 in the morning in a cold sweat about what we did, but we had to do it.” (Participant A6) Participants expressed concern that the health system may be on the brink of a staff mental health crisis and the risk of a staff “exodus” (Participant A6) from the healthcare service. One participant commented that the current level of sick leave by staff “has gone through the roof” (Participant A3), with an increase in staff reporting “mental health issues” (Participant A3). Concern was expressed that senior staff within the health service did not appreciate the work done locally by management and staff to respond to the situation. Participants also noted a desire for further acknowledgement of the resilience, altruism, and adaptability displayed by healthcare staff. Collaborative practices: a central influence for supporting system resilience Collaborative practices: a central influence for supporting system resilience The cyber-attack resulted in the flattening of the healthcare hierarchy with shared decision-making evident at local levels. This heightened autonomy led to an empowered frontline workforce. One participant described an “equal platform”, “hierarchy was gone” (Participant C6), which facilitated staff to speak up: “Yeah, and people were in pushing ideas, you know? So that there was a good yeah kind of collaboration of people, saying can we try this? and you know, in many cases the people on the floor as always had the solution.” (Participant A10) Participants reflected positively on how staff came together as a team, with communication playing a critical role in the success of the cyber-attack response. When asked what the key learning had been from this experience, one participant responded: “teamwork, teamwork, teamwork and communication.” (Participant A10). While communication underpinned the success of mitigations introduced, staff understood the need for flexibility due to the evolving situation: “There would have been an update as to where we were that morning and OK that could have changed by evening and, if necessary, there was an evening meeting and then that was fed back to I suppose the staff on the ground.” (Participant A14) “There would have been an update as to where we were that morning and OK that could have changed by evening and, if necessary, there was an evening meeting and then that was fed back to I suppose the staff on the ground.” (Participant A14) However, while effective collaboration at local level made staff feel empowered to respond, participants identified several gaps in top-down communication during the cyber-attack. Communication nationally to local services was described as “far from superior” (Participant M4). There was frustration that the protracted nature of the disruption to health services was not communicated to the public: “whether it wasn't communicated or they weren't picking up on it in the media” (Participant A12), leaving local management feeling unsupported. There was also significant concern that the clinical risk posed by the removal of systems was not understood at the national level: Page 7/16 Page 7/16 “I don't think there was the understanding there that our clinical risk was so great because we had absolutely no histories. Collaborative practices: a central influence for supporting system resilience All of the other hospitals had paper charts, whereas in the acutes and everywhere, we had absolutely nothing” (Participant M4) “I don't think there was the understanding there that our clinical risk was so great because we had absolutely no histories. All of the other hospitals had paper charts, whereas in the acutes and everywhere, we had absolutely nothing” (Participant M4) However, participants working on teams with local staff members attending national meetings were more positive about top-down communication, describing these links as “valuable” (Participant A6). Immediate and long-term consequences requiring action Gaps in IT Infrastructure & Support The cyber-attack has drawn attention to the lack of investment in IT infrastructure across all services. Participants explained how they had expressed concern about poor ICT systems over long periods of time, with one senior staff member working within community services commenting that “we need to do something serious nationally in our approach to IT” (Participant C1), while another senior staff member, also working within community services, commented: Page 8/16 Page 8/16 “There is a big gap if you look at a comparable organisation in the private sector - like their IT systems or their access is light years ahead of ours.” (Participant C7) While ICT systems are more advanced in the acute and maternity services, participants across these services also expressed concern about lack of investment. Old ICT systems in use throughout the health system are more vulnerable to cyber-attack, with one participant working in a laboratory setting commenting that “we are Windows 7 based and we'd be hoping to move to Windows 10” and further commented that “the speed at which the IT is replaced within the HSE is a big problem” (Participant A13). There was also a general perception that there is a lack of planning to address this deficit nationally: “planning for that nationally, it doesn't seem to be there” (Participant A13). Participants reported a lack of trust in the ability of IT systems to protect against future events. While some services had backup systems in place, these backups were designed for shorter ICT outages and due to the protracted nature of the event, the backups quickly became ineffectual. Staff expressed frustration with IT support, “I don’t think we have enough IT support locally or nationally, it’s all extremely frustrating” (Participant C1). This frustration particularly related to the support provided through national helpdesks, but participants also mentioned delays in equipment procurement and set-up of new starters which all adversely impacted care delivery in the aftermath of the cyber-attack: “the service level is bad to start with, but it’s way worse since the cyber-attack” (Participant C1). Participants felt local IT staff on-site were needed to support their systems. Gaps in IT Infrastructure & Support The teams that had close relationships with their local IT support found it beneficial in understanding the emerging situation throughout the cyber-attack: “there's a ICT department in X hospital which would be involved in PC and hardware replacement and we would have close ties and liaise with them and discuss any national information that they might have fed through their chief and would have a close link in with their line manager as well, so we were all working very closely to hope to try and get the best possible outcomes as quickly as possible, but also as safely as possible each way along, each step along the road”. Participant A13 On-going and future concerns Focus groups were conducted 4 to 5 months after the cyber-attack, yet all participants noted that the effects of the cyber-attack were on-going. Performance issues with core systems and lack of remote access to computers for some staff has impacted productivity: “You know everybody thinks we're back to normal and actually a week later it kind of dropped off the news and stuff, but we were really suffering and we continue…our productivity is very poor still from a digital point of view [...]…so yeah, that's our estimate. Yeah, 30% productivity down on the digital side still”. (Participant A9) Participants commented that cyber-security measures employed in response to the cyber-attack also resulted in systems becoming slower, less accessible, with an increase in equipment downtime as Page 9/16 Page 9/16 external support providers, such as engineers, could no longer remotely access equipment in many instances. Staff were also concerned that the cyber-attack impacted the trust of patients and service-users. The adverse impact of the cyber-attack on waiting lists, already problematic due to the impact of the pandemic, added a further challenge to staff striving to deliver a quality service for patients: “We have been trying in our hospital to work on waiting lists and try and improve the quality and the timeliness of the service that we provide to our patients. COVID impacted on this, but the cyber-attack impacted even more on it and there's nothing out there for our patients.” (Participant M3) Clinical staff also suspected that they will not be fully aware of the impact of mitigations on patients treated during this period for a long time to come. Manual workarounds developed were prone to risks such as redundancy, missing data, and retrospective data entry and reconciliation. A number of participants were concerned that risks and incidents may emerge in future: “It's a huge burden that people have to carry because we don't know what wasn't done. So, we can only hope that we captured all of the patients that weren't seen that need to be seen. We don't…We can't be certain. We don't have any kind of procedures in place to be able to follow this through, which is a very unnerving place to be when you're responsible for the health of a patient and particularly where time can be of grave importance in terms of outcomes”. On-going and future concerns (Participant M3) Staff wanted reassurance that the health system has learnt from this experience and will be able to better protect staff and patients from the impact of a similar event should it occur in the future. One participant working in an acute setting asked “what has been put in place nationally to help protect us from this and protect our patients from something similar” and further stated “because it’s likely it’s going to come again, we need to be more prepared”. (Participant A7). Discussion Psychological safety is important in healthcare, particularly in challenging times. This study finds that staff felt empowered to speak-up, respond to the challenges and introduce local adaptions to ensure continuity of service. This is reflective of a service that prioritises patient safety, a supportive environment, familiarity with colleagues, and the flattened hierarchy (14). Transparent communication and good collaboration are both key strategies to building a resilience health system (15). Psychological safety promotes collaborative practices, which in turn supports quality and safe care (16). Staff collaborated and recognised the importance of teamwork and effective communication needed to introduce the necessary mitigations. An independent report carried out to determine the facts surrounding the cyber-attack (6) noted the dedication and effort by individuals at all levels from across the HSE, impacted hospitals, CHOs, and third parties all going “above and beyond” in their call of duty in response to this incidence. The report noted that, in times of significant challenge or emergencies, staff in the health services are resilient, respond quickly, and have an ability to implement actions and workarounds to maintain even a basic continuity of service to their patients. However, the effect the cyber-attack had on those working within the Irish health system cannot be underestimated. While the resilience, dedication, and innovation of staff ensured necessary services remained open, the findings further comment that this environment put additional stress on a system of healthcare professionals that were already exhausted by four waves of the COVID-19 pandemic. While the pandemic has severely exacerbated workplace stress for healthcare workers worldwide (17), participants in this study felt the stress placed on staff by the attack was more severe than the cumulative effect of the pandemic. This stress was further amplified by a lack of public awareness about the impact and duration of the cyber-attack. While there was no evidence that healthcare provision in the immediate aftermath of the attack has resulted in harm to patients, many staff carry the burden of worry over clinical decisions made and the potential loss of patient information during this period. Healthcare quality is the key outcome for resilience in healthcare (18). Healthcare quality includes clinical effectiveness, patient safety, timeliness, patient centeredness, care coordination, efficiency, and equity (19), and each of these dimensions was compromised during the cyber-attack. Discussion While determinations on health system resilience often focus on surges in demand due to natural disasters and disease outbreaks, shocks that do not directly impact demand but compromise the ability of a national system of healthcare to deliver core services are rarely explored. The ransomware cyber- attack in May 2021 on the national ICT infrastructure of the public health system in Ireland resulted in the widespread removal of access to ICT systems across all public health services. Healthcare services are especially vulnerable to cyber-attacks due to the nature of services, where loss of access to electronic health records, radiology, and pathology results can have a devastating impact on patient safety (12, 13). In the absence of ICT systems, staff within the Irish health service and allied service providers rapidly focused on developing and implementing manual workarounds to ensure continuity of services and maintain patient safety. Experienced staff assumed leadership roles and offered reassurance and guidance to colleagues who had no experience operating outside of a digital environment. The Page 10/16 Page 10/16 knowledge and experience of these staff members ensured that risks to patient safety were minimised (8). Psychological safety is important in healthcare, particularly in challenging times. This study finds that staff felt empowered to speak-up, respond to the challenges and introduce local adaptions to ensure continuity of service. This is reflective of a service that prioritises patient safety, a supportive environment, familiarity with colleagues, and the flattened hierarchy (14). Transparent communication and good collaboration are both key strategies to building a resilience health system (15). Psychological safety promotes collaborative practices, which in turn supports quality and safe care (16). Staff collaborated and recognised the importance of teamwork and effective communication needed to introduce the necessary mitigations. An independent report carried out to determine the facts surrounding the cyber-attack (6) noted the dedication and effort by individuals at all levels from across the HSE, impacted hospitals, CHOs, and third parties all going “above and beyond” in their call of duty in response to this incidence. The report noted that, in times of significant challenge or emergencies, staff in the health services are resilient, respond quickly, and have an ability to implement actions and workarounds to maintain even a basic continuity of service to their patients. knowledge and experience of these staff members ensured that risks to patient safety were minimised (8). Ethics and consent to participate The study was in accordance with Declaration of Helsinki.The study received ethical approval from the Clinical Research Ethics Committee of the Cork Teaching Hospitals (ECM 4 (s) 6/7/2021 & ECM 3 (oo) 10/08/2021). All participants provided written informed consent and all potentially identifiable characteristics were removed from each transcript to maintain anonymity. Discussion For example, equity, patient centredness, and timeliness were all compromised by the health systems inability to withstand this attack and to resume normal service speedily, further increasing already long waiting lists. Resilience must be designed within a health system. Robust, self-regulating health systems need investment in the so-called slow variables, ones that take a long time to change but are required to construct a stable platform for health care delivery (4). National leadership, a committed workforce and sufficient infrastructure are key to building resilience (20). Preparedness is key for resilience (15), yet there is little evidence that building resilience was a priority within the Irish health system. There was no assessment of system capacities and weaknesses, and no investment in vulnerable components of the system (6). Resilient performance is achieved through a combination of absorption of challenges, adaptation and transformation to continue operations in the face of disruptions (21). Those working within the Irish health system absorbed the challenge of the response to the cyber-attack and introduced adaptions to keep services functioning, at least at a basic level. However, adaptions were largely temporary, and Page 11/16 Page 11/16 transformation could not occur due to the system’s lack of readiness for such an event. The dedication, innovation, and commitment of health workers propped-up the health system at this time of crisis. This is resilience by happenstance rather than design (4). transformation could not occur due to the system’s lack of readiness for such an event. The dedication, innovation, and commitment of health workers propped-up the health system at this time of crisis. This is resilience by happenstance rather than design (4). Conclusion The cyber-attack on the Irish health system in May 2021 resulted in the widespread removal of access to ICT systems. While services including radiology, diagnostics, maternity, and oncology were prioritised for reinstatement, recovery efforts continued for over four months, a timescale far greater that initially expected. The absence of the appropriate capacity within the health system infrastructure to protect patients, service users, and staff from the impact of such an attack, meant this lack of system resilience was compensated for by a resilient workforce. Within the context of the prevailing COVID-19 pandemic, this was an enormous burden on a dedicated workforce. The adverse impact of this attack may have long-term and far-reaching consequences for the wellbeing of staff. Design and investment in a resilient health system must be prioritised. Limitations Focus groups explored the experiences of staff working in acute, maternity, and community settings during the cyber-attack, and all participants were drawn from a specific region. While a broad range of staff from a variety of services participated, their experiences may not reflect those of staff working within other regions of the national public health service. Furthermore, this study does not include the experiences of health service staff working at a national level, including those working within IT, who were greatly impacted by the cyber-attack. This study was conducted in September/October 2021, shortly after the cyber-attack, and does not reflect staff experiences of operational and infrastructural IT system improvements subsequently introduced. Funding No funding was provided for this research. Acknowledgements The study team wishes to express their sincere thanks to the participants of this study who generously gave their time to share insights and learning to inform the findings. We would also like to thank all HSE staff who assisted us in the participant recruitment process and facilitated us in setting-up the online focus group sessions. We would like to thank the wider team involved in the supporting mixed-method study including Michael Carton, Grainne Cosgrove, Fiona Culkin, Loretto Grogan, Catherine Hogan, Emma Hogan, Loretta Jenkins and Margaret McGarry. Consent for publication Not applicable. Authors’ contributions Conceptualization (OH, GM, ZK, TMD), Data curation (GM, ZK), Formal analysis (TMD, LR), Interpretation (TMD, LR, GM, ZK), Methodology (TMD, LR, GM, ZK), Writing – original draft (TMD, LR, GM, ZK), Writing – review and editing (OH,TMD, LR, GM, ZK). The authors read and approved the final manuscript. Availability of data and materials The datasets generated and analysed during the current study are not publicly available as ethical approval was granted on the basis that the anonymised transcripts would be held by the HSE for 2 years then securely destroyed. Page 12/16 Competing interests OH and GM are employees of the Health Service Executive. OH is the National Clinical Director of the Directorate and GM is responsible for Qualitative Research and Evaluation. The remaining authors, TMD, LR and ZK, declare no competing interests. References Qualitative Res 11. Braun V, Clarke V. Using thematic analysis in psychology. Qualitative Res Psychol. 2006;3(2):77–101. 12. Argaw ST, Bempong N-E, Eshaya-Chauvin B, Flahault A. The state of research on cyberattacks against hospitals and available best practice recommendations: a scoping review. BMC Med Inf Decis Mak. 2019;19(1):1–11. 13. Ghafur S, Kristensen S, Honeyford K, Martin G, Darzi A, Aylin P. 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https://openalex.org/W2078831742	https://gmd.copernicus.org/articles/8/151/2015/gmd-8-151-2015.pdf	English	null	Implementation and comparison of a suite of heat stress metrics within the Community Land Model version 4.5	Geoscientific model development	2,015	cc-by	15,818	Correspondence to: J. R. Buzan (jonathan.buzan@unh.edu) Received: 21 June 2014 – Published in Geosci. Model Dev. Discuss.: 8 August 2014 Revised: 2 December 2014 – Accepted: 23 December 2014 – Published: 5 February 2015 Received: 21 June 2014 – Published in Geosci. Model Dev. Discuss.: 8 August 2014 Revised: 2 December 2014 – Accepted: 23 December 2014 – Published: 5 February 2015 Abstract. We implement and analyze 13 different metrics (4 moist thermodynamic quantities and 9 heat stress metrics) in the Community Land Model (CLM4.5), the land surface component of the Community Earth System Model (CESM). We call these routines the HumanIndexMod. We limit the al- gorithms of the HumanIndexMod to meteorological inputs of temperature, moisture, and pressure for their calculation. All metrics assume no direct sunlight exposure. The goal of this project is to implement a common framework for calculating operationally used heat stress metrics, in climate models, of- ﬂine output, and locally sourced weather data sets, with the intent that the HumanIndexMod may be used with the broad- est of applications. The thermodynamic quantities use the lat- est, most accurate and efﬁcient algorithms available, which in turn are used as inputs to the heat stress metrics. There are three advantages of adding these metrics to CLM4.5: (1) im- proved moist thermodynamic quantities; (2) quantifying heat stress in every available environment within CLM4.5; and (3) these metrics may be used with human, animal, and in- dustrial applications. culation and the improved wet bulb calculation are ±1.5 ◦C. These differences are important due to human responses to heat stress being nonlinear. Furthermore, we show heat stress has unique regional characteristics. Some metrics have a strong dependency on regionally extreme moisture, while others have a strong dependency on regionally extreme tem- perature. 1 Introduction Heat-related conditions are the number one cause of death from natural disaster in the United States – more than tor- nadoes, ﬂooding, and hurricanes combined (NOAAWatch, 2014). Short-term duration (hours) of exposure to heat while working may increase the incidence of heat exhaustion and heat stroke (Liang et al., 2011). However, long-term exposure (heat waves or seasonally high heat), even without working, may drastically increase morbidity and mortality (Kjellstrom et al., 2009a). Although there is high uncertainty in the num- ber of deaths, the 2003 European heat wave killed 40 000 people during a couple weeks in August (García-Herrera et al., 2010), and tens of thousands more altogether for the en- tire summer (Robine et al., 2008). The 2010 Russian heat wave, the worst recorded heat wave, killed 55 000 people over the midsummer (Barriopedro et al., 2011). We demonstrate the capabilities of the HumanIndexMod in a default conﬁguration simulation using CLM4.5. We out- put 4× daily temporal resolution globally. We show that the advantage of implementing these routines into CLM4.5 is capturing the nonlinearity of the covariation of temperature and moisture conditions. For example, we show that there are systematic biases of up to 1.5 ◦C between monthly and ±0.5 ◦C between 4× daily ofﬂine calculations and the on- line instantaneous calculation, respectively. Additionally, we show that the differences between an inaccurate wet bulb cal- A growing literature is concerned with the frequency and duration of heat waves (Seneviratne et al., 2012, and ref- erences therein). One study concluded that intensiﬁcation of 500 hPa height anomalies will produce more severe heat Implementation and comparison of a suite of heat stress metrics within the Community Land Model version 4.5 J. R. Buzan1,2, K. Oleson3, and M. Huber1,2 1Department of Earth Sciences, University of New Hampshire, Durham, New Hampshire, USA 2Earth Systems Research Center, Institute for the Study of Earth, Ocean, and Space, University of New Hampshire, Durham, New Hampshire, USA 3 3National Center for Atmospheric Research, Boulder, Colorado, USA Correspondence to: J. R. Buzan (jonathan.buzan@unh.edu) J. R. Buzan et al.: Implementation and comparison of a suite of heat stress metrics Benestad’s (2011) mis- application implies that most humans should die every year, because a great portion of the world reaches temperatures of 35 ◦C for more than a 6 h period. There are a limited number of studies validating, explor- ing, or using heat stress metrics on a global scale (Kjell- strom et al., 2009b; Hyatt et al., 2010; Sherwood and Hu- ber, 2010; Fischer and Schar, 2010; Fischer et al., 2012; Fis- cher and Knutti, 2012; Willett and Sherwood, 2012; Dunne et al., 2013; Kjellstrom et al., 2013; Oleson et al., 2013b). Algorithms for measuring heat stress and labor capacity are based upon sub-daily rates of exposure to heat stress (Par- sons, 2006). Most of these studies do not capture the diur- nal cycle of heat stress (Kjellstrom et al., 2009b; Hyatt et al., 2010; Fischer and Schar, 2010; Fischer and Knutti, 2012; Willett and Sherwood, 2012; Dunne et al., 2013; Kjellstrom et al., 2013), thus not representing either nighttime highs or daytime extremes. Only one study includes solar radiation as a component in heat stress (Kjellstrom et al., 2013). Differ- ent metrics are used between each study, and only one study attempts to compare more than two metrics (Oleson et al., 2013b). Our goal here is to improve the situation by creating a module that calculates a large suite of metrics, using the most accurate and efﬁcient algorithms available, that may be used with as many applications as possible: climate models, of- ﬂine archive data, model validation studies, and weather sta- tion data sets. We call this module the HumanIndexMod. The module calculates four moist thermodynamic quantities and nine heat stress metrics. These heat stress metrics are in op- erational use worldwide and cover a wide range of assump- tions. Various forms of moist thermodynamic calculations (Buck, 1981; Davies-Jones, 2008; Stull, 2011) and heat stress metrics are criticized for their inaccuracies (Budd, 2008; Al- fano et al., 2012; Davies-Jones, 2008). Buck’s (1981) moist thermodynamic calculations are not as accurate as those of Bolton (1980) yet are used in a recent study (Kjellstrom et al., 2013). Wet bulb temperature calculations are different between multiple studies (Hyatt et al., 2010; Sherwood and Huber, 2010; Dunne et al., 2013; Kjellstrom et al., 2013; Oleson et al., 2013b). Hyatt et al. (2010) and Kjellstrom et al. (2013) use natural wet bulb temperature (e.g. J. R. Buzan et al.: Implementation and comparison of a suite of heat stress metrics 152 waves over Europe and North America in the future (Meehl and Tebaldi, 2004). Another study shows that, even with including the global warming “hiatus”, there is an increas- ing occurrence of extreme temperatures (Seneviratne et al., 2014). Multiple studies associate lack of precipitation and/or low soil moisture with contributing to high temperatures (Fischer et al., 2007; Mueller and Seneviratne, 2012; Mi- ralles et al., 2014). per for their calculation of wet bulb temperature. However, Sherwood and Huber’s (2010) wet bulb temperature calcu- lations use Bolton’s (1980) equivalent potential temperature Eq. (38), rather than the more accurate Eq. (39) (Bolton, 1980; Davies-Jones, 2008, 2009). Furthermore, their calcu- lation is only valid for wet bulb temperatures above 10 ◦C. Dunne et al. (2013), on the other hand, use wet bulb po- tential temperature Eq. (3.4) in Davies-Jones (2008), yet the recommended equations for wet bulb potential temperature are Eqs. (3.5)–(3.7) and (3.8) (Davies-Jones, 2008) for the temperature ranges used in their paper. Dunne et al. (2013) also use Bolton’s (1980) equivalent potential temperature Eq. (40), rather than the more accurate Eq. (39) (Bolton, 1980). Additionally, Dunne et al. (2013) use a variation of WBGT that is heavily criticized, the indoorWBGT (Budd, 2008). Regarding humans, however, temperature differences are not the primary method for heat dissipation. Evaporation of sweat is crucial to maintaining homeostasis, and none of the aforementioned studies incorporate atmospheric moisture to measure heat stress. Many diagnostic and prognostic meth- ods were developed to diagnose heat stress (over a 100-year history, Table 1) – such as the Wet Bulb Globe Temperature (WBGT), the Discomfort Index (DI), or Heat Index (HI) – and policy makers have decided to incorporate these indices in weather warning systems (Epstein and Moran, 2006; Par- sons, 2006, 2013; Rothfusz, 1990; Fiala et al., 2011). Occasionally, results using heat stress limits are misin- terpreted. One study confuses wet bulb temperature thresh- olds with dry bulb temperature thresholds (Benestad, 2011). This has misleading consequences as their results do not in- clude moisture metrics, yet the author cites Sherwood and Huber’s (2010) wet bulb threshold (35 ◦C) as the threshold value for their temperature analysis. The wet bulb tempera- ture at 35 ◦C is a theoretical limit where humans would die from heat stress after 6 h of exposure. Published by Copernicus Publications on behalf of the European Geosciences Union. Published by Copernicus Publications on behalf of the European Geosciences Union. J. R. Buzan et al.: Implementation and comparison of a suite of heat stress metrics Brake, 2001) – a calculation that, due to nonlinear components of its equa- tion, may have multiple steady-state solutions (Alfano et al., 2010). Oleson et al. (2013b) uses a recent formulation of wet bulb temperature that is limited in effective range of input temperatures (Stull, 2011) (we go into further detail on this equation in Sect. 2). Sherwood and Huber (2010) and Dunne et al. (2013) both use Davies-Jones (2008) as their source pa- As an example of numerous applications, we imple- ment the HumanIndexMod into the Community Land Model (CLM4.5), a component model of the Community Earth Sys- tem Model (CESM), maintained by the National Center for Atmospheric Research (NCAR) (Hurrell et al., 2013). The metrics are directly calculated at the sub-grid scale, capturing heat stress in every environment: urban areas, lakes, vegeta- tion, and bare ground. We show examples of the advantages of calculating these metrics at the model time step as com- pared to lower temporal resolution, and the importance of using accurate moist thermodynamic calculations. We also show that having all metrics calculated at the same time al- lows for comparison of metrics between each other, and al- lows for unique analysis of conditional distributions of the inputs. Finally, we show that the metrics may also be used as model diagnostics. www.geosci-model-dev.net/8/151/2015/ Geosci. Model Dev., 8, 151–170, 2015 www.geosci-model-dev.net/8/151/2015/ J. R. Buzan et al.: Implementation and comparison of a suite of heat stress metrics Table 1. Heat stress diagnostics and prognostic models. Metric Type Ref. Wet bulb temperature Temperature Haldane (1905) Effective Temperature Index Houghton and Yaglou (1923) Equivalent temperature Temperature Dufton (1929) Heat Stress Index Index Belding and Hatch (1955) Wet Bulb Globe Temperature Index Yaglou and Minard (1957) Discomfort Index Index Thom (1959) Temperature Humidity Index Index Ingram (1965) Temp. regulation in man Prognostic Stolwijk and Hardy (1966) Physiological mathematical model Prognostic Wyndham and Atkins (1968) Solar heat in man Index Breckenridge and Goldman (1971) Mathematical model temperature in man Prognostic Stolwijk (1971) New Effective Temperature Index Gagge (1972) Humidex Index Masterson and Richardson (1979) Sultriness Index Index Steadman (1979a) Mathematical model thermal regulation Prognostic Stolwijk (1980) Apparent Temperature Index Steadman (1984) Heat Index Index Rothfusz (1990) Computer-based thermal response Prognostic Haslam and Parsons (1994) SCENARIO Prognostic Kraning and Gonzalez (1997) Computer model human thermo-regulation Prognostic Fiala et al. (1999, 2001) PET Index Höppe (1999) Environmental Stress Index Index Moran et al. J. R. Buzan et al.: Implementation and comparison of a suite of heat stress metrics (2001) SCENARIO Monte Carlo Prognostic Gonzalez (2004) Generalized transient thermal model Prognostic Khan et al. (2004) ISO 7243 WBGT Index Parsons (2006) IDCA Prognostic Yokota et al. (2008) Physiological Equivalent Temperature Index Jendritzky et al. (2009) UTCI Index Fiala et al. (2010) UTCI-Fiala model Index-Prognostic Fiala et al. (2011) Index of Equivalent Temperature Index Liang et al. (2011) 153 J. R. Buzan et al.: Implementation and comparison of a suite of heat stress metrics The outline of the paper is as follows: Sect. 2 (Heat stress modeling) focuses on the development, calculation, and use of these 13 metrics. Section 3 (Methods) describes the imple- mentation and model setup. Section 4 (Results) presents the results of a model simulation using these metrics. Section 5 (Discussion) discusses the implications of the research, and Sect. 6 (Summary) presents the conclusions of the paper. paper. Each index that we chose uses a combination of atmo- spheric variables: temperature (T ), humidity (Q), and pres- sure (P). We chose these metrics because they are in oper- ational use globally by industry, governments, and weather services. Furthermore, these metrics may be applied to the broadest range of uses: climate and weather forecasting mod- els, archive data sets, and local weather stations. , , Sections 2.2–2.4 describe the metrics that we have cho- sen to implement in the HumanIndexMod (see variables de- ﬁned in Table 2). Most of the metrics have units of tem- perature, which may be misleading. The metrics have tem- perature scales for comparative purposes only, as the met- rics are an index, not a true thermodynamic quantity. We break these metrics into three categories, based upon design philosophies: comfort, physiological response, and empirical ﬁt. Comfort-based algorithms are a quantiﬁcation of behav- ioral or “feels-like” reactions to heat in both animals and hu- mans. Physiological indices quantify the physical response mechanisms within a human or animal, such as changes in heart rate or core temperatures. The empirical indices quan- tify relationships between weather conditions and a non- Geosci. Model Dev., 8, 151–170, 2015 2.2 Comfort algorithms HI = −42.379 + 2.04901523TF + 10.14333127RH+ −0.22475541TFRH + −6.83783 × 10−3T 2 F + −5.481717 × 10−2RH2 + 1.22874 × 10−3T 2 F RH + 8.5282 × 10−4TFRH2 + −1.99 × 10−6T 2 F RH2 (3) We use Apparent Temperature, Heat Index, Humidex, and Temperature Humidity Index for Comfort to account for comfort level. These metrics were either tailored to the global locations where they were developed or streamlined for ease of use from physiology models. The underlying philosophical approach to deriving comfort metrics is repre- senting behavioral reactions to levels of comfort (Masterson and Richardson, 1979; Steadman, 1979a). The goal of these equations of comfort is to match the levels of discomfort to appropriate warnings for laborers (Gagge et al., 1972) and livestock (Renaudeau et al., 2012). Discomfort in humans sets in much earlier than physiological responses; i.e., the human body provides an early warning to the mind that con- tinuing the activity may lead to disastrous consequences. For example, when heat exhaustion sets in, the body is sweating profusely, and often there are symptoms of dizziness. How- ever, the actual core temperature for heat exhaustion is de- ﬁned at 38.5 ◦C, which is considerably lower than heat stroke (42 ◦C). We describe the four comfort-based algorithms be- low. (3) Here, air temperature (TF) and HI are in degrees Fahren- heit. HI has a number of assumptions. The equation assumes a walking person in shorts and T-shirt, who is male and weighs ∼147 lbs (Rothfusz, 1990). Additionally, this subject is not in direct sunlight. As with AT, HI represents a feels-like temperature, based upon levels of discomfort. HI uses a scale for determining heat stress: 27–32 ◦C is caution, 33–39 ◦C is extreme caution, 40–51 ◦C is danger, and ≥52 ◦C is extreme danger. Humidex (HUMIDEX) was developed for the Meteoro- logical Service of Canada and describes the feels-like tem- perature for humans (Masterson and Richardson, 1979). The original equation used dew point temperature, rather than speciﬁc humidity. The equation was modiﬁed to use vapor pressure, instead: HUMIDEX = TC + 5 9 eRH 100 −10 . (4) (4) Apparent Temperature was developed using a combination of wind, radiation, and heat transfer to measure thermal com- fort and thermal responses in humans (Steadman, 1994). AT is used by the Australian Bureau of Meteorology and was de- veloped for climates in Australia (ABM, 2014). 2.2 Comfort algorithms The metric is an approximation of a prognostic thermal model of human comfort (Steadman 1979a, b, 1984). HUMIDEX is unitless because the authors recognized that the index is a measure of heat load. The index has a series of thresholds: 30 is some discomfort, 46 is dangerous, and 54 is imminent heat stroke (Masterson and Richardson, 1979). HUMIDEX is unitless because the authors recognized that the index is a measure of heat load. The index has a series of thresholds: 30 is some discomfort, 46 is dangerous, and 54 is imminent heat stroke (Masterson and Richardson, 1979). The Temperature Humidity Index for Comfort (THIC) is a modiﬁcation of the Temperature Humidity Index (THI) (In- gram, 1965). Comfort was quantiﬁed for livestock through THIC (NWSCR, 1976). We use the original calibration, which is for pigs (Ingram, 1965). The index is unitless: AT = TC + 3.3eRH 1000 −0.7u10 m −4, (1) eRH = RH 100, esPa, (2) (1) (2) where the vapor pressure (eRH) is in pascals and is calculated from the relative humidity (RH in %) and saturated vapor pressure (esPa, also in pascals). We use this notation because es (Table 2) is in millibars. These variable names are the explicit names of the variables in the HumanIndexMod. AT uses the wind velocity (m s−1) measured at the 10 m height (u10 m). Air temperature (TC) and AT are in units of degrees Celsius. AT is the only metric in the HumanIndexMod that includes an explicit calculation for wind velocity; the other metrics assume a reference wind. We included this metric due to a previously used legacy version within CLM4.5 (Ole- son et al., 2013b). An assumption made by AT is that the sub- ject is outside but not exposed to direct sunlight. AT has no explicit thresholds; rather, the index shows an ampliﬁcation of temperatures. Previous work, however, has used tempera- ture percentiles to describe AT (Oleson et al., 2013b). where the vapor pressure (eRH) is in pascals and is calculated from the relative humidity (RH in %) and saturated vapor pressure (esPa, also in pascals). We use this notation because es (Table 2) is in millibars. These variable names are the explicit names of the variables in the HumanIndexMod. AT uses the wind velocity (m s−1) measured at the 10 m height (u10 m). Air temperature (TC) and AT are in units of degrees Celsius. J. R. Buzan et al.: Implementation and comparison of a suite of heat stress metrics a heat stress early warning system, and the index was created as a polynomial ﬁt to Steadman’s (1979a) comfort model (Rothfusz, 1990). a heat stress early warning system, and the index was created as a polynomial ﬁt to Steadman’s (1979a) comfort model (Rothfusz, 1990). physical or comfort-related attribute. For example, an empir- ical algorithm’s result may determine how much work may be completed per hour per weather condition. 2.1 Background The primary focus of this paper is on atmospheric-variable- based heat stress metrics that we introduce into the Hu- manIndexMod. The models for determining heat stress for humans vary greatly, ranging from simple indices to com- plex prognostic physiology modeling (Table 1). Prognostic thermal models are beyond the scope of this paper, as they require more than atmospheric inputs. Additionally, metrics that include radiation and wind (with one exception: Appar- ent Temperature, or AT) are also beyond the scope of this Geosci. Model Dev., 8, 151–170, 2015 www.geosci-model-dev.net/8/151/2015/ 154 2.2 Comfort algorithms AT is the only metric in the HumanIndexMod that includes an explicit calculation for wind velocity; the other metrics assume a reference wind. We included this metric due to a previously used legacy version within CLM4.5 (Ole- son et al., 2013b). An assumption made by AT is that the sub- ject is outside but not exposed to direct sunlight. AT has no explicit thresholds; rather, the index shows an ampliﬁcation of temperatures. Previous work, however, has used tempera- ture percentiles to describe AT (Oleson et al., 2013b). THIC = 0.72Tw + 0.72TC + 40.6, (5) (5) where wet bulb temperature (Tw) is in degrees Celsius. The index is used to describe behavioral changes in large animals due to discomfort (seeking shade, submerging in mud, etc.). The index is in active use by the livestock industry for local heat stress and future climate considerations (Lucas et al., 2000; Renaudeau et al., 2012). The index describes qualita- tive threat levels for animals: 75 is alert, 79–83 is dangerous, and 84+ is very dangerous. There are different approaches to the development of THIC, including considerations of phys- iology of large animals. 2.4 Empirical algorithms Berglund and Yokota, 2005; Gribok et al., 2008; Maloney and Forbes, 2011; Havenith et al., 2011; Gonzalez et al., 2012; Chan et al., 2012). Most metrics require radiation mea- surements or heart rates, and/or even sweat rates. The avail- able metrics that are calibrated for physiological responses using only meteorological inputs, however, are limited, such as the Temperature Humidity Index for Physiology (THIP; Ingram, 1965): The last category of metrics is derived from ﬁrst principle thermo-physiology models, or changes in worker productiv- ity, etc., and then reduced by empirical ﬁt. The ﬁrst metric we present is a widely used modiﬁcation of an industry la- bor standard, the simpliﬁed Wet Bulb Globe Temperature (sWBGT): ity, etc., and then reduced by empirical ﬁt. The ﬁrst metric we present is a widely used modiﬁcation of an industry la- bor standard, the simpliﬁed Wet Bulb Globe Temperature (sWBGT): sWBGT = 0.56TC + 0.393eRH 100 + 3.94. (7) (6) THIP = 0.63Tw + 1.17TC + 32. (6) (7) THIP and THIC are modiﬁcations of the THI. Addition- ally, THIC and THIP have applications beyond heat stress. THIP and THIC threshold levels are computed from both in- door and outdoor atmospheric variables. The differences be- tween outdoor and indoor values are used to evaluate evapo- rative cooling mechanisms, e.g., swamp coolers (Gates et al., 1991a, b). sWBGT was designed for estimating heat stress in sports medicine and adopted by the Australian Bureau of Meteorol- ogy; however, it is acknowledged that its accuracy of repre- senting the original labor industry index may be questionable (ABOM, 2010; ACSM, 1984, 1987). We chose, however, to implement sWBGT due to its wide use. sWBGT is unitless, and its threat levels are as follows: 26.7–29.3 is green, or alert; 29.4–31.0 is yellow, or caution; 31.1–32.1 is red, or potentially dangerous; and ≥32.2 is black, or dangerous con- ditions (US Army, 2003). www.geosci-model-dev.net/8/151/2015/ 2.3 Physiology algorithms Numerous metrics are based upon direct physiological re- sponses within humans and animals; however, almost all of them are complicated algorithms (e.g., Moran et al., 2001; Heat Index was developed using a similar process to AT. The United States National Weather Service (NWS) required Geosci. Model Dev., 8, 151–170, 2015 www.geosci-model-dev.net/8/151/2015/ J. R. Buzan et al.: Implementation and comparison of a suite of heat stress metrics Table 2. Moist temperature variables and heat stress metrics. Metric Variable Equation no. Temperature (kelvin) T n/a Temperature (Celsius) TC n/a Temperature (Fahrenheit) TF n/a Pressure P n/a Relative humidity RH n/a Speciﬁc humidity Q n/a 10 m winds u10 m n/a Vapor pressure (mb) eRH 2 Vapor pressure (Pa) esPa n/a Saturated vapor pressure (mb) es (A13) Derivative saturated vapor pressure des/dT (A16) Log derivative saturated vapor pressure d(ln(es))/dT (A15) Mixing ratio rs (A14) Derivative mixing ratio drs/dT (A17) Function of equivalent potential temperature f (θE) (A18) Derivative of function of equivalent potential temperature f ′(θE) (A20) Wet bulb temperature Tw (A22) Wet bulb temperature, Stull TwS (8)–(9) Lifting condensation temperature TL (A2) Moist potential temperature θDL (A3) Equivalent potential temperature θE (A4) Equivalent temperature TE (A5) Heat Index HI (3) Apparent Temperature AT (1) Humidex HUMIDEX (4) Simpliﬁed WBGT sWBGT (7) Discomfort Index DI (10) Temperature Humidity Index for Comfort THIC (5) Temperature Humidity Index for Physiology THIP (6) Swamp cooler efﬁciency 65 % SWMP65 (11) Swamp cooler efﬁciency 80 % SWMP80 (11) 155 J. R. Buzan et al.: Implementation and comparison of a suite of heat stress metrics 3 Methods −20 < TC < 50 −2.27TC + 27.7 < RH < 99 (9) (9) Our approach is to choose a subset of heat stress metrics that are in common use operationally by governments and/or used extensively in prior climate modeling studies (Table 3). We do this in order to provide a framework to allow compar- isons of metrics across studies, and we designate the algo- rithms the HumanIndexMod. Section 3.1 describes CLM4.5. Section 3.2 discusses the implementation of the HumanIn- dexMod into CLM4.5. Section 3.3 describes our simulation setup that we use to demonstrate the capabilities of the Hu- manIndexMod. The simulation is for showcasing the Hu- manIndexMod, not as an experiment for describing real cli- mate or climate change. Section 3.4 describes a unique ap- plication method for analyzing heat stress. We compute DI with both TwS and Tw calculated using our implementation of Davies-Jones (2008) (Eq. A22). We keep the legacy version (Stull, 2011) for comparative purposes. DI is calculated from these inputs: DI = 0.5Tw + 0.5TC, (10) (10) where the DI is unitless and the values are an indicator of threats to the populations: 21–24 is < 50 % of population in discomfort, 24–27 > 50 % of population in discomfort, 27– 29 most of the population in discomfort, 29–32 severe stress, and > 32 is state of emergency (Giles et al., 1990). The last index we present is a measurement of the capac- ity of evaporative cooling mechanisms. Often, these are re- ferred to as swamp coolers. Large-scale swamp coolers gen- erally work by spraying a “mist” into the air, or blowing air through a wet mesh. This mist then comes in contact with the skin, and subsequently evaporates, thus cooling down the subject. In dry environments, they can be an effective mass cooling mechanism. Unfortunately, swamp coolers raise the local humidity considerably, reducing the effectiveness of di- rect evaporation from the skin. Swamp coolers are measured by their efﬁciency: 3.1 The structure of Community Land Model version 4.5 We use CLM version 4.5, which was released in June 2013 (Oleson et al., 2013a). Boundary conditions for CLM4.5 con- sist of land cover and atmospheric weather conditions. Each grid cell in CLM4.5 can include vegetation, lakes, wetlands, glacier, and urban areas. There are new parameterizations and models for snow cover, lakes, and crops; a new biogeo- chemical cycles model; and new urban classiﬁcations (Ole- son et al., 2013a). The urban biome, a single-layer canyon model, is designed to represent the “heat island”, where tem- peratures are ampliﬁed by urban environments (Oleson et al., 2008a, b, 2010a, b, 2011). The heat-island effect can increase the likelihood of complications from human heat stress (Ole- son, 2012). η = TC −Tt TC −Tw 100 %, (11) (11) where η(%) is the efﬁciency, and Tt is the target temperature for the room to be cooled towards in degrees Celsius (Koca et al., 1991). Rearranging Eq. (11) and solving for Tt, J. R. Buzan et al.: Implementation and comparison of a suite of heat stress metrics 156 with two different efﬁciencies: SWMP80, for η at 80 %, and SWMP65 for η at 65 %. With the mist-injected air cooled to Tt, Tt is approximately equal to a new local Tw. Humid en- vironments or environments that are hot and have an above- average RH relative to their normally high T severely limit the cooling potential of swamp coolers. The livestock indus- try uses evaporative cooling mechanisms for cooling, often in conjunction with THIP and THIC, as mentioned previ- ously (Gates et al., 1991a, b). Due to their low cost, swamp coolers are used throughout the world as a method of cool- ing buildings and houses. No one has implemented SWMP65 and SWMP80 in global models, and we believe that this will provide many uses to industry by its inclusion in CLM4.5. Table 2 shows which metrics are discussed in this paper. Discomfort Index was developed in the 1950s as a cali- bration for air conditioners (Thom, 1959). It was adapted by the Israeli Defense Force as a decision-making tool regarding heat stress (Epstein and Moran, 2006). DI requires Tw and TC. The computation of Tw in the past was computationally expensive, and the DI equations often used approximations (e.g., Oleson et al., 2013b): with two different efﬁciencies: SWMP80, for η at 80 %, and SWMP65 for η at 65 %. With the mist-injected air cooled to Tt, Tt is approximately equal to a new local Tw. Humid en- vironments or environments that are hot and have an above- average RH relative to their normally high T severely limit the cooling potential of swamp coolers. The livestock indus- try uses evaporative cooling mechanisms for cooling, often in conjunction with THIP and THIC, as mentioned previ- ously (Gates et al., 1991a, b). Due to their low cost, swamp coolers are used throughout the world as a method of cool- ing buildings and houses. No one has implemented SWMP65 and SWMP80 in global models, and we believe that this will provide many uses to industry by its inclusion in CLM4.5. Table 2 shows which metrics are discussed in this paper. TwS = TCarctan 0.151977 √ RH + 8.313659 + arctan(TC + RH) −arctan(RH −1.676331) + 0.00391838RH3/ 2arctan(0.023101RH) −4.68035, (8) (8) where TwS is the wet bulb temperature in degrees Celsius (Stull, 2011). Stull’s function has limited range of effective accuracy. www.geosci-model-dev.net/8/151/2015/ Geosci. Model Dev., 8, 151–170, 2015 J. R. Buzan et al.: Implementation and comparison of a suite of heat stress metrics 3.2 HumanIndexMod design and implementation Tt = TC −η 100 (TC −Tw), (12) (12) (12) There are two philosophical aspects to the design of the HumanIndexMod: (1) accurate and efﬁcient moist thermo- dynamic algorithms, and (2) a modular format to increase use through both narrowly focused applications and broadly based studies. The module is in an open-source format and is where Tt is now the predicted temperature based upon en- vironmental variables. The maximum efﬁciency of typical swamp coolers is 80 %, and a typical value of a sub-standard mechanism is 65 % (Koca et al., 1991). Thus, we calculate Tt www.geosci-model-dev.net/8/151/2015/ Geosci. Model Dev., 8, 151–170, 2015 J. R. Buzan et al.: Implementation and comparison of a suite of heat stress metrics J. R. Buzan et al.: Implementation and comparison of a suite of heat stress metrics 157 J. R. Buzan et al.: Implementation and comparison of a suite of heat stress metrics 157 Table 3. List of previous heat stress studies. Studies using data sets, reanalysis, and/or model output that range from ∼1900 until ∼2010 are labeled “modern” and from ∼2005 to ∼2100 are labeled “future”. Some studies do not analyze heat stress quantitatively (Assessment). Table 3. List of previous heat stress studies. Studies using data sets, reanalysis, and/or model output that range from ∼1900 until ∼2010 are labeled “modern” and from ∼2005 to ∼2100 are labeled “future”. Some studies do not analyze heat stress quantitatively (Assessment). Table 3. List of previous heat stress studies. Studies using data sets, reanalysis, and/or model output that range from ∼1900 until ∼2010 are labeled “modern” and from ∼2005 to ∼2100 are labeled “future”. Some studies do not analyze heat stress quantitatively (Assessment). Location Metric Time Model Ref. Mediterranean Sea HI Modern and future RegCM3 Diffenbaugh et al. (2007) Delhi WBGT Modern NOAA Kjellstrom et al. (2009a) World sWBGT Future HadCM3 Kjellstrom et al. (2009b) World cities WBGT, T Modern and future NOAA/various models Kjellstrom et al. (2009c) Global PET variation Future ECHAM4 Jendritzky and Tinz (2009) Global Tw Modern and future CCSM3/ERA Interim Sherwood and Huber (2010) Europe HI, HUMIDEX Future ENSEMBLES Fischer and Schar (2010) Global indoorWBGT Modern and future NOAA Hyatt et al. (2010) Global – Modern Assessment Nilsson and Kjellstrom (2010) Southern Brazil UTCI Modern Direct measurement Bröde et al. (2012) Global sWBGT Modern and future CLM4 Fischer et al. (12) (2012) Global sWBGT Modern and future HadCRUH/ISD-NCDC Willett and Sherwood (2012) Global T Modern Various data sets SREX IPCC (2012) Western India WBGT, T Modern Direct measurement Nag et al. (2013) California farms – Modern Assessment Stoecklin-Marois et al. (2013) Thailand – Modern Assessment Tawatsupa et al. (2013) Nepal sWBGT, HI, HUMIDEX Modern Direct measurement Pradhan et al. (2013) Southeast Asia WBGT Modern and future GSOD/CRU/BCM2 Kjellstrom et al. (2013) Quebec T Future Assessment Adam-Poupart et al. (2013) Global indoorWBGT Modern and future ESM2M/NCEP-NCAR Dunne et al. (2013) United States sWBGT, DI, HI, HUMIDEX, AT Modern and future CLM4/CLMU/WRF Oleson et al. (2013b) incorporated into the CLM4.5 developer branch (the module itself is available from the corresponding author’s website). The modular format encourages adapting the code to spe- ciﬁc needs, whether that focus is on moist thermodynamics or heat stress. The inclusion of heat stress metrics covering comfort, physiology, and empirical philosophies encourages the use of HumanIndexMod for many applications. and it is not available in QSatMod. Additionally, QSat_2 cal- culates f (θE) (Eq. A18) with respect to the input temperature and the subsequent derivatives. These are required to cal- culate Tw (Eq. A22) using Davies-Jones (2008) and cannot be accomplished using QSatMod. We show acceptable dif- ferences between the Stull version of wet bulb temperature (TwS) calculated using both QSatMod and QSat_2 (Fig. 1a). The new subroutines improve CLM4.5 by calculating previ- ously unused thermodynamic quantities. Additionally, these routines are useful moist thermodynamic routines for other data sets for researchers to use, thus expanding the capacity of the HumanIndexMod. We directly implemented the code into the CLM4.5 ar- chitecture through seven modules. Four of these modules – BareGroundFuxesMod, CanopyFluxesMod, SlakeFluxes- Mod, and UrbanMod – call the HumanIndexMod. The HumanIndexMod is calculated for every surface type in CLM4.5. The design of CLM4.5 allows the urban and rural components, where the rural component represents the nat- ural vegetation surface, to be archived separately for inter- comparison. The HumanIndexMod uses the 2 m calculations of water vapor, temperature, and pressure, as well as 10 m winds. Three other modules are modiﬁed with the implemen- tation process. These modules – clmtype, clmtypeInitMod, and histFldsMod – are used for initializing memory and out- putting variable history ﬁles. We implement speciﬁc thermodynamic routines developed by Davies-Jones (2008) to accurately calculate Tw (see Ap- pendix A). Geosci. Model Dev., 8, 151–170, 2015 J. R. Buzan et al.: Implementation and comparison of a suite of heat stress metrics J. R. Buzan et al.: Implementation and comparison of a suite of heat stress metric 158 J. R. Buzan et al.: Implementation and comparison of a suite of heat stress metrics Wet Bulb Evaluation Difference (K) Tw - TwS_QSat_2 TwS_QSat_2 - TwS_QSatMod Tw_Monthly - Tw_Instantaneous Tw_4xDaily - Tw_Instantaneous Difference (K) Difference (K) Difference (K) T (K) T (K) T (K) T (K) (a) (b) (d) (c) on of wet bulb temperatures. The boxes represent the 90 % conﬁdence interval. The upper and lower tails represent the interval. The horizontal line in each box is the median value. (a) is the difference between TwS using QSat_2 saturated d QSatMod saturated vapor pressure over the valid range for TwS. (b) is the difference between Tw (Davies-Jones, 2008) 11) (both using QSat_2 saturated vapor pressure calculation) over the valid range for TwS. (c) is the difference between hly averaged input ﬁelds and model instantaneous ﬁelds to calculate monthly Tw. (d) is the difference between using model input ﬁelds and model instantaneous ﬁelds to calculate 4× daily Tw. For (a), (b), and (d) the inputs of T , P, and Q are el 4× daily ﬁelds from the years 2001–2010. For (c) the inputs of T , P , and Q are derived from model monthly ﬁelds from 10. Wet Bulb Evaluation Wet Bulb Evaluation Wet Bulb Evaluation Difference (K) Tw - TwS_QSat_2 TwS_QSat_2 - TwS_QSatMod Difference (K) T (K) T (K) (a) (b) TwS_QSat_2 - TwS_QSatMod Difference (K) Difference (K) Tw_Monthly - Tw_Instantaneous Tw_4xDaily - Tw_Instantaneous Tw_Monthly - Tw_Instantaneous Tw_4xDaily - Tw_Instantaneous Difference (K) Difference (K) T (K) T (K) (d) (c) Figure 1. Evaluation of wet bulb temperatures. The boxes represent the 90 % conﬁdence interval. The upper and lower tails represent the 100 % conﬁdence interval. The horizontal line in each box is the median value. (a) is the difference between TwS using QSat_2 saturated vapor pressure and QSatMod saturated vapor pressure over the valid range for TwS. (b) is the difference between Tw (Davies-Jones, 2008) and TwS (Stull, 2011) (both using QSat_2 saturated vapor pressure calculation) over the valid range for TwS. (c) is the difference between using model monthly averaged input ﬁelds and model instantaneous ﬁelds to calculate monthly Tw. (d) is the difference between using model 4× daily averaged input ﬁelds and model instantaneous ﬁelds to calculate 4× daily Tw. 3.3 CLM4.5 experimental setup tion rate, temperature, speciﬁc humidity, zonal and merid- ional winds, and surface pressure. Our simulation has the carbon and nitrogen cycling on (biogeophysics “CN”). The simulation was initialized at year 1850, on a ﬁnite volume grid of 1◦× 1◦, using boundary conditions provided from NCAR (Sam Levis, personal com- munication, 2013). The simulation spun up while cycling 3 times over CRUNCEP 1901–1920 forcings. Once completed, our experiment used the spun-up land conditions and ran the entirety of 1901–2010. CLM4.5 may be executed independently of the other mod- els in CESM; this is called an I-compset. To do so, CLM4.5 requires atmospheric boundary conditions. We use the de- fault data set for CLM4.5 – CRUNCEP. CRUNCEP is the NCEP/NCAR reanalysis product (Kalnay et al., 1996) cor- rected and downscaled by the Climatic Research Unit (CRU) gridded observations data set from the University of East Anglia (Mitchell and Jones, 2005). The time period is 4× daily from 1901 to 2010, and it is on a regular grid of ∼0.5◦× 0.5◦. The combination of CRU and NCEP prod- ucts was to correct for biases in the reanalysis product and improve overall resolution (Casado et al., 2013). To drive CLM4.5 we used surface solar radiation, surface precipita- J. R. Buzan et al.: Implementation and comparison of a suite of heat stress metrics For (a), (b), and (d) the inputs of T , P, and Q are derived from model 4× daily ﬁelds from the years 2001–2010. For (c) the inputs of T , P , and Q are derived from model monthly ﬁelds from the years 2001–2010. 3.4 Heat stress indices analysis An open question is what drives extreme high-heat-stress events, which are, by deﬁnition, rare events. For example, we Geosci. Model Dev., 8, 151–170, 2015 (12) Equation (A4) is the most accurate and efﬁcient θE calculation available (Bolton, 1980; Davies-Jones, 2009). Calculating Eq. (A4) required implementing TL and θDL (Eqs. A2 and A3, respectively) into the HumanIndexMod. T , P , and Q from CLM4.5 are used to calculate θE and TE (Eq. A5). TE, a quantity used in a previous heat stress study (Fischer and Knutti, 2012), is an input into QSat_2 for cal- culating the initial guess of Tw, and subsequently followed by the accelerated Newton–Raphson method (Eqs. A9–A22). We found it advantageous to split the heat stress quantities into their own subroutines, allowing the user to choose which quantities are to be calculated. The minimum requirements to execute the entire module are T (K), P (Pa), RH (%), Q (g kg−1), e (Pa), and u10 m (m s−1). Table 4 shows the sub- routines, input requirements, and outputs in HumanIndex- Mod. Moist thermodynamic water vapor quantities in CLM4.5 are calculated within QSatMod. We use the outputs from QSatMod as the inputs to the HumanIndexMod. Within the HumanIndexMod, we created a subroutine, QSat_2, which has all the same functionalities as QSatMod. This subroutine uses the August–Roche–Magnus (ARM) equation (Eq. A13) rather than the Flatau et al. (1992) polynomial equations for vapor pressure in QSatMod. The log derivative of ARM (Eq. A15) is a critical component of the calculation of Tw, Geosci. Model Dev., 8, 151–170, 2015 www.geosci-model-dev.net/8/151/2015/ www.geosci-model-dev.net/8/151/2015/ www.geosci-model-dev.net/8/151/2015/ J. R. Buzan et al.: Implementation and comparison of a suite of heat stress metrics 1 Table 4. The HumanIndexMod: subroutine names, required inputs, and variables calculated. Name Subroutine Input Calculates Moist thermodynamics Wet_Bulb T,eRH, P , RH, Q TE,θE,Tw Wet bulb temperature, Stull Wet_BulbS TC, RH TwS Heat Index HeatIndex TC, RH HI Apparent Temperature AppTemp TC,eRH, Wind AT Simpliﬁed WBGT swbgt TC,eRH sWBGT Humidex hmdex TC,eRH HUMIDEX Discomfort Index dis_coi TC,Tw DI Discomfort Index w/Stull dis_coiS TC,TwS DI Temperature Humidity Index THIndex TC,Tw THIC, THIP Swamp cooler efﬁciency SwampCoolEff TC,Tw SWMP65, SWMP80 Kelvin to Celsius KtoC T TC Vapor pressure VaporPres RH, es eRH Saturated vapor pressure QSat_2 T,P es, des/dT , d(ln(es))/dT , rs, drs/dT , f (θE), f ′(θE) J. R. Buzan et al.: Implementation and comparison of a suite of heat stress metrics 159 cannot determine from the mean climate state or from theory, in a warm and humid climate, whether abnormally high tem- perature, abnormally high moisture, or a combination of the two caused a heat stress event. This is a question of the co- variance of perturbations of temperature and humidity, not a statement of mean conditions, and there is no theory to ex- plain these situations. For example, we may apply Reynolds averaging to the NWS Heat Index equation (Eq. 3): 40 150 6-hour intervals, ∼10 038 days) and 95th percentiles (hottest 8030 6-hour intervals, ∼2008 days). Every 6 h period that exceeds the percentiles was located within the time series, and we calculated the conditional dis- tributions. For example, the 99th percentile exceedance of HI isolated the top 1606 hottest time steps for each grid cell. Af- ter isolating these time steps, we use this distribution as a mask to isolate all other quantities (e.g., temperature and hu- midity), allowing cross comparison between all metrics and HI. The goal is to develop an analysis technique comparing all covariances of the metrics within CLM4.5. HI = a + bT + cRH + dT RH + eT 2 + f RH2 + gT 2RH + hT RH2 + iT 2RH2 + h dRH′T ′ + eT ′2 + f RH′2 + gT ′2RH′ +hT ′RH′2 + iT ′2RH′2i , (13) After the conditional distributions are calculated, we, again, compute the statistical dispersion (mean, variance, ex- ceedance, etc.) of the percentiles. We display this analysis with maps in two ways. www.geosci-model-dev.net/8/151/2015/ (1) We show the exceedance value of a metric, and (2) we show T −Q regime plots of that same metric. We calculate the T −Q regimes through expected rank values (Fig. 2). This required a series of steps. (1) We take the conditional distribution of T and Q that represent exceedance percentile of the source heat stress or moist ther- modynamic metric. (2) We take the expected value (median) of the conditional distributions of T and Q and determine what percentile they come from in their respective time se- ries. (3) We condition these values on each other to create the expected rank values (Fig. 2). (13) where a, b, c, d, e, f , g, h, and i are constants in the poly- nomial. RH and T are relative humidity and temperature, respectively. We are not concerned with the terms outside the brackets, as they are the means. The terms within the bracket are representative of turbulent effects on the heat index, which we are discussing. It is these turbulent states where a GCM is able to determine these individual factors, by calculating the heat stress metrics and thermodynamic quantities at every model time step. Furthermore, each heat stress metric has different assumptions (such as body size, physical ﬁtness, etc.) that weight temperature and humidity differently. A high-heat-stress event indicated by one metric does not necessarily transfer onto another metric. where a, b, c, d, e, f , g, h, and i are constants in the poly- nomial. RH and T are relative humidity and temperature, respectively. We are not concerned with the terms outside the brackets, as they are the means. The terms within the bracket are representative of turbulent effects on the heat index, which we are discussing. It is these turbulent states where a GCM is able to determine these individual factors, by calculating the heat stress metrics and thermodynamic quantities at every model time step. Furthermore, each heat stress metric has different assumptions (such as body size, physical ﬁtness, etc.) that weight temperature and humidity differently. A high-heat-stress event indicated by one metric does not necessarily transfer onto another metric. 4 Results We present a snap shot of the many metrics calculated. First, we present results of our evaluation the improved moist ther- modynamic calculations and the implementation these met- rics into CLM4.5 (Fig. 1). Second, we show an example of the possible global applications for these metrics (Figs. 3– 6). This approach characterizes heat stress within CLM4.5 in response to one observation reanalysis product, the CRUN- CEP. Thus, we outputted 4× daily averages of the heat stress metrics and the corresponding surface pressure (P), 2 m tem- perature (T ), 10 m winds (u10 m), and 2 m humidity (Q) ﬁelds. We computed statistics for the time series (mean, variance, exceedance, etc.). We focus primarily on the 99th percentiles (hottest 1606 6-hour intervals, ∼402 days) but also show some of the robust features with the 75th (hottest Geosci. Model Dev., 8, 151–170, 2015 J. R. Buzan et al.: Implementation and comparison of a suite of heat stress metrics 160 <66.6 66.6<83.3 83.3<90 90<99 ≥99 <66.6 Extreme T 66.6<83.3 83.3<90 90<99 ≥99 Extreme Q Conditioned T Percentile Conditioned Q Percentile Figure 2. Expected value ranking. T and Q conditioned upon ex- ceedance value of a heat stress or moist thermodynamic metric. The T and Q values are compared to their respective time series as a percentile. These T and Q percentiles are binned and are compared to each other. Extreme Q are greens and extreme T are magentas. <66.6 66.6<83.3 83.3<90 90<99 ≥99 <66.6 Extreme T 66.6<83.3 83.3<90 90<99 ≥99 Extreme Q Conditioned T Percentile Conditioned Q Percentile c). sWBGT shows values exceeding minimum metric warn- ing levels (e.g., China, northern Africa), whereas HI does not have necessarily the same warning. The 95th percentile shows that moderate levels of heat stress have expanded into higher latitudes (Fig. 4a–c). At equatorial and monsoonal re- gions, heat stress labor reductions should be in effect as it is not safe to work outside and, in some cases (western Africa, the Arabian Peninsula, and the Himalayan wall), not safe to work at all. At the 99th percentile, severe heat stress is expe- rienced in the monsoonal regions (Fig. 5a–c). These maxima correlate with maxima in Tw (Fig. 5c). g The T −Q regime maps show that partitioning of heat stress into T and Q begins in regional locations at the 75th percentile (Fig. 3d–f). The partitioning occurs in low lati- tudes and is not consistent between metrics. At the 95th per- centile, the partitioning expands into higher latitudes; how- ever, many areas (continental interiors) remain equally de- pendent on T and Q (Fig. 4d–f). Tw is largely driven by extreme moisture (Fig. 4f) and in some locations (mon- soonal Africa, Indian sub-continent, and equatorial South America) very extreme moisture. HI is driven by T (Fig. 4e), and sWBGT is mixed between extreme Q and extreme T (Fig. 4d). All three metrics agree with T in the western United States and Middle East. At the 99th percentile, HI, although dominated by T worldwide, shows sign reversals in very small locations (Fig. 5e). Extreme Q expands for Tw, and all of the low latitudes experience moisture depen- dence except for the western United States and Middle East (Fig. 5f). sWBGT has some reversal of T - to Q-dominated heat stress (western Africa). Q largely expands worldwide. J. R. Buzan et al.: Implementation and comparison of a suite of heat stress metrics In all instances, except for HI, high latitudes are equally de- pendent on Q and T for heat stress. Figure 2. Expected value ranking. T and Q conditioned upon ex- ceedance value of a heat stress or moist thermodynamic metric. The T and Q values are compared to their respective time series as a percentile. These T and Q percentiles are binned and are compared to each other. Extreme Q are greens and extreme T are magentas. 4.1 Evaluation of improved moist thermodynamic quantities We present a series of box-and-whisker plots demonstrating the value added of implementing (1) accurate and efﬁcient moist thermodynamic quantities, and (2) online calculation of the heat stress metrics is an improvement over calculat- ing these metrics using monthly or 4× daily model output (Fig. 1). Figure 1a shows the difference in the Stull (2011) wet bulb temperature calculated using the saturated vapor pressure from Davies-Jones (2008) (QSat_2) and Flatau et al. (1992) (QSatMod). The differences are minimal. How- ever, our point is that the Davies-Jones (2008) method for wet bulb temperature is preferred. We show the difference between wet bulb temperatures using Stull (2011) calcu- lated with QSat_2 and Davies-Jones (2008) (which requires QSat_2) (Fig. 1b). Differences are greater than 1 K between Stull (2011) and Davies-Jones (2008) methods, and they are temperature dependent (Fig. 1b). Lastly, we show the difference between calculating Davies-Jones (2008) Tw us- ing monthly and 4× daily averaged model data versus the model instantaneous calculations (Fig. 1c and d, respec- tively). Using model-averaged data instead of the instanta- neous data systematically overestimates Tw by more than 1 K for monthly and 0.5 K for 4× daily output. Our ﬁnal maps show SWMP65, SWMP80, and θE at the 99th percentile. Maxima for θE are spatially the same as Tw (Figs. 5c and 6c). Additionally, θE partitions towards Q, just as Tw shows (Figs. 5f and 6f). Spatial patterns between SWMP65 and HI are similar (Figs. 5b and 6a), and their regime maps show similar partitioning toward T globally, ex- cept for select locations of strong monsoonal locations that show Q dependency (Figs. 5e and 6d). Lastly, SWMP80 and sWBGT share similar spatial patterns (Figs. 5a and 6b). As with the other paired metrics, their T −Q regime maps share the same characteristics (Figs. 5d and 6e). Low lati- tudes show strong Q dependence, and higher latitudes switch to a T dependence. www.geosci-model-dev.net/8/151/2015/ www.geosci-model-dev.net/8/151/2015/ J. R. Buzan et al.: Implementation and comparison of a suite of heat stress metrics 161 sWBGT unitless HI °C Tw °C 22 23 14 30 35 22 38 47 30 (a) (b) (c) (d) (e) (f) Regime Metric 75th Percentile Extreme T Extreme Q Extreme T Extreme Q Extreme T Extreme Q Figure 3. 75th percentile exceedance value of three metrics for (a) sWBGT, (b) HI, and (c) Tw (left). Expected rank value T −Q regime maps (d), (e), and (f) (right) conditioned by (a), (b) and (c), respectively. Rank values for (d)–(f) are described in Fig. 2. Figure 3. 75th percentile exceedance value of three metrics for (a) sWBGT, (b) HI, and (c) Tw (left). Expected rank value T −Q regime maps (d), (e), and (f) (right) conditioned by (a), (b) and (c), respectively. Rank values for (d)–(f) are described in Fig. 2. (Fig. 1c, d). Even using daily or sub-daily averages (Kjell- strom et al., 2009b; Hyatt et al., 2010; Fischer and Schar, 2010; Fischer and Knutti, 2012; Willett and Sherwood, 2012; Kjellstrom et al., 2013) potentially overestimates heat stress. This is due to the nonlinear covariance of T and Q, and aver- ages miss these extremes. Ultimately, capturing the diurnal cycle is crucial for quantifying heat stress extremes (Ole- son et al., 2013b). Heat-stress-related illness is exacerbated by high-heat-stress nights as well as daytimes. To accurately calculate these extremes, one needs either high-temporal- resolution data or direct computation of them at each time step within climate models. We discuss the results from the implementation separately: moist thermodynamics and heat stress. climate models and reanalysis fall along constant lines of TE (Eq. A5), even out to the 99th percentile of daily values (Fis- cher and Knutti, 2012). The spread between models is small as compared to the spread in T ; thus using heat stress met- rics in Earth system modeling may reduce the uncertainties of climate change (Fischer and Knutti, 2012). Previous modeling studies have demonstrated that urban equatorial regions transition to a nearly permanent high-heat- stress environment when considering global warming (Fis- cher et al., 2012; Oleson et al., 2013b). The convective re- gions are areas with the highest heat stress maximums and are often near coastal locations. Many of these metropoli- tan areas are in monsoonal regions, which have strong yearly moisture variability, yet the partitioning of heat stress is to- wards Q, not T , in these regions (Figs. 5d–f and 6d–f). J. R. Buzan et al.: Implementation and comparison of a suite of heat stress metrics Heat stress in both equatorial and monsoonal regions is expected to increase dramatically when considering global warming (Kjellstrom et al., 2009b; Fischer and Knutti, 2012; Dunne et al., 2013; Oleson et al., 2013b). Accurate moist thermody- 4.2 Exceedance values and regime maps We designed the HumanIndexMod to calculate diagnostic heat stress and moist thermodynamics systematically. There are many approaches to evaluating heat stress. Monthly and seasonal temperature and moisture averages were used for general applications (Dunne et al., 2013); however these averages overestimate the potential severity of heat stress We show exceedance and T −Q regime maps for the 75th and 95th percentiles of three metrics, and 99th percentiles of six metrics. The maps show spatial patterns of heat stress and characteristics. Equatorial and monsoonal regions show moderate levels of heat stress in the 75th percentile (Fig. 3a– Geosci. Model Dev., 8, 151–170, 2015 www.geosci-model-dev.net/8/151/2015/ www.geosci-model-dev.net/8/151/2015/ J. R. Buzan et al.: Implementation and comparison of a suite of heat stress metrics J. R. Buzan et al.: Implementation and comparison of a suite of heat stress metric 162 sWBGT unitless HI °C Tw °C 22 23 14 30 35 22 38 47 30 (a) (b) (c) (d) (e) (f) Regime Metric 95th Percentile Extreme T Extreme Q Extreme T Extreme Q Extreme T Extreme Q Figure 4. 95th percentile exceedance value of three metrics for (a) sWBGT, (b) HI, and (c) Tw (left). Expected rank value T −Q regime maps (d), (e), and (f) (right) conditioned by (a), (b) and (c), respectively. Rank values for (d)–(f) are described in Fig. 2. sWBGT unitless HI °C Tw °C 22 23 14 30 35 22 38 47 30 (a) (b) (c) (d) (e) (f) Regime Metric 95th Percentile Extreme T Extreme Q Extreme T Extreme Q Extreme T Extreme Q Fi 4 95 h il d l f h i f ( ) WBGT (b) HI d ( ) T (l f ) E d k l T Q i Regime Figure 4. 95th percentile exceedance value of three metrics for (a) sWBGT, (b) HI, and (c) Tw (left). Expected rank value T −Q regime maps (d), (e), and (f) (right) conditioned by (a), (b) and (c), respectively. Rank values for (d)–(f) are described in Fig. 2. namic calculations from the HumanIndexMod will aid future characterizations of heat stress. ple, the original equation from which sWBGT was derived was calibrated using US Marine Corps Marines during basic training (Minard et al., 1957), who are in top physical con- dition. HI was calibrated for an “average” American male (Steadman, 1979a; Rothfusz, 1990). Calculating these heat stress metrics, and the many others in the HumanIndexMod, at every time step within climate models was previously in- tractable due to insufﬁcient data storage capabilities for high- temporal-resolution variables. We show that SWMP65 and SWMP80 diverge in their values (Fig. 6a, b and d, e). Yet, SWMP80 and sWBGT are similar in spatial patterns and regimes, while HI and SWMP65 have similar patterns and regimes. What links SWMP65 and SWMP80 together is Tw. Swamp coolers are evaporators, and, as their efﬁciency ap- proaches 100 %, their solutions approach Tw. Figures 5 and 6 are similar to a circuit resistor, or stomatal resistance (Oke, 1987), which is measure of efﬁciency. The average person (HI) may be acting as a stronger resistor to evaporation than one who is acclimatized (sWBGT). J. R. Buzan et al.: Implementation and comparison of a suite of heat stress metrics The HumanIndexMod may explore the effects of acclimatization and its impact on 5.1 Moist thermodynamics The spatial distributions of high heat stress are robust be- tween CLM model versions (Oleson et al., 2011, 2013b; Fis- cher et al., 2012). Due to the conservation of energy and en- tropy, calculating moist thermodynamic variables shows that Geosci. Model Dev., 8, 151–170, 2015 www.geosci-model-dev.net/8/151/2015/ J. R. Buzan et al.: Implementation and comparison of a suite of heat stress metrics 163 sWBGT unitless HI °C Tw °C 22 23 14 30 35 22 38 47 30 (a) (b) (c) (d) (e) (f) Extreme T Regime Metric 99th Percentile Extreme Q Extreme Q Extreme T Extreme Q Extreme T Figure 5. 99th percentile exceedance value of three metrics for (a) sWBGT, (b) HI, and (c) Tw (left). Expected rank value T −Q regime ( ) ( ) (f) ( ) ( ) ( ) ( ) (f) sWBGT unitless HI °C Tw °C 22 23 14 30 35 22 38 47 30 (a) (b) (c) (d) (e) (f) Extreme T Regime Metric 99th Percentile Extreme Q Extreme Q Extreme T Extreme Q Extreme T Figure 5. 99th percentile exceedance value of three metrics for (a) sWBGT, (b) HI, and (c) Tw (left). Expected rank value T −Q regime maps (d), (e), and (f) (right) conditioned by (a), (b) and (c), respectively. Rank values for (d)–(f) are described in Fig. 2. Regime (f) Extreme Q 30 Extreme T Figure 5. 99th percentile exceedance value of three metrics for (a) sWBGT, (b) HI, and (c) Tw (left). Expected rank value T −Q regime maps (d), (e), and (f) (right) conditioned by (a), (b) and (c), respectively. Rank values for (d)–(f) are described in Fig. 2. efﬁciency of evaporative cooling through climate modeling. This type of research may ultimately reduce the number met- rics required for computing heat stress. Less complicated models represent the human body as a sin- gle cylinder with multiple layers (Kraning and Gonzalez, 1997). Neither computational method is currently coupled to Earth system models, and this is a signiﬁcant gap in deter- mining future heat stress impacts that the HumanIndexMod may not be able to fulﬁll. To make progress towards repre- senting the effects of heat stress on the human body prognos- tically, we recommend, as a ﬁrst step, incorporating mean radiant temperature of humans. Radiation is a major compo- nent of human energy balance, and implementing this also allows incorporating more accurate diagnostics, such as Wet Bulb Globe Temperature (Minard et al., 1957) and the Uni- versal Thermal Climate Index (Havenith et al., 2012). Exposure to high temperatures and moisture, ultimately, threatens humans physically, and long-term exposure may lead to death. Extreme moist temperatures are projected to increase in the future, and potentially may reach deadly ex- tremes, permanently in some regions (Sherwood and Huber, 2010). J. R. Buzan et al.: Implementation and comparison of a suite of heat stress metrics Heat stress indices have the ability to diagnose in- stantaneous exposure. Diagnostic models, however, cannot measure or evaluate the potential impacts of long-term expo- sure to heat stress accurately. Prognostic thermal physiolog- ical models can be used to predict the complexities of heat stress on humans. Prognostic thermal physiology considers wind, ambient temperature, and moisture from the environment, as well as internal processes, such as blood ﬂow and sweat. There are numerous different forms of prognostic models (Table 1). Some of them are quite complicated, using hundreds of grid cells to represent all parts of the body (Fiala et al., 1999). 5.2 Heat stress We show that there are two regimes of heat stress globally in agreement between metrics in the CRUNCEP CLM4.5 sim- ulation, T (western United States and Middle East) and Q (monsoonal regions). The western United States and Mid- dle East regions consistently have higher temperatures and lower humidities than the monsoonal areas. However, we show that maximum heat stress is partitioned between T and Q globally. Characterizing arid regions versus non-arid re- gions may require different heat stress metrics (e.g., Oleson et al., 2013b; speciﬁcally the comparison between Phoenix and Houston). The HumanIndexMod provides this capabil- ity. The assumptions/calibrations that derived the heat stress metrics in the HumanIndexMod are another avenue of re- search that may be explored using a global model. For exam- Geosci. Model Dev., 8, 151–170, 2015 www.geosci-model-dev.net/8/151/2015/ www.geosci-model-dev.net/8/151/2015/ J. R. Buzan et al.: Implementation and comparison of a suite of heat stress metrics 164 SWMP65 °C SWMP80 °C θe °C 14 14 50 22 22 86 30 30 121 (a) (b) (c) (d) (e) (f) Extreme T Regime Metric 99th Percentile Extreme Q Extreme Q Extreme T Extreme Q Extreme T Figure 6. 99th percentile exceedance value of three metrics for (a) SWMP65, (b) SWMP80, and (c) θE (left). Expected rank value T −Q regime maps (d), (e), and (f) (right) conditioned by (a), (b) and (c), respectively. Rank values for (d)–(f) are described in Fig. 2. 30 121 Figure 6. 99th percentile exceedance value of three metrics for (a) SWMP65, (b) SWMP80, and (c) θE (left). Expected rank value T −Q regime maps (d), (e), and (f) (right) conditioned by (a), (b) and (c), respectively. Rank values for (d)–(f) are described in Fig. 2. curate and efﬁcient algorithms available. The heat stress met- rics cover three developmental philosophies: comfort-, phys- iologically, and empirically based algorithms. The code is designed, with minimal effort, to be implemented into gen- eral circulation, land surface, and weather forecasting mod- els. Additionally, this code may be used with archived data formats and local weather stations. radiation. Radiation is a non-negligible component of heat stress. As a consequence, the heat stress metrics presented always assume that the subject is not in direct solar exposure. Additionally, the indices represent a diagnostic environment for heat stress. These metrics do not incorporate prognos- tic components or complex physiology of the human thermal system. Overall, the HumanIndexMod provides a systematic way for implementing an aspect of thermo-animal physiology into an Earth system modeling framework. Incorporating the HumanIndexMod into a variety of different models would provide a baseline for model–model comparisons of heat stress, such as the Coupled Model Intercomparison Project (CMIP) (Taylor et al., 2012) and other collaborative model- ing frameworks. We encourage researchers to incorporate the HumanIndexMod into their research environments. Furthermore, we have implemented the HumanIndexMod into the latest public release version of CLM4.5. Archival is ﬂexible, as the user may choose to turn on high-frequency output, and the default is monthly averages. Additionally, monthly urban and rural output of the metrics is default. We show that the module may be used to explore new avenues of research: characterization of human heat stress, model di- agnostics, and intercomparisons of heat stress metrics. Geosci. Model Dev., 8, 151–170, 2015 6 Summary We present the HumanIndexMod, which calculates nine heat stress metrics and four moist thermodynamical quantities. The moist thermodynamic variables use the latest, most ac- www.geosci-model-dev.net/8/151/2015/ Geosci. Model Dev., 8, 151–170, 2015 164 J. R. Buzan et al.: Implementation and comparison of a suite of heat stress metrics J. R. Buzan et al.: Implementation and comparison of a suite of heat stress metrics 165 Appendix A: Moist thermodynamics hot, (A10) cold = 0 : 1 ≤ C TE λ ≤D (π) = 1 , (A11) hot = 1 : TE > 355.15 = 0 , (A12) Tw = k1(π) −1.21cold −1.45hot− (k2(π) −1.21cold) C TE λ + 0.58 C TE λ ! hot, (A10) (A10) cold = 0 : 1 ≤ C TE λ ≤D (π) = 1 , (A11) hot = 1 : TE > 355.15 = 0 , (A12) (A11) θDL = T p0 p −e κd T TL 0.00028r , (A3) (A3) (A12) where e is the parcel vapor pressure (mb) (using CLM4.5, this is the 2 m vapor pressure), and r is the mixing ratio (g kg−1) (this is converted from the 2 m height Q to r in CLM4.5). Third, the parcel is raised to a great height where all latent heat is transferred to the air parcel, and the water is rained out, giving the solution to θE. There are many methods for representing this process. The analytical solution (Holton, 1972) is computationally prohibitive in atmospheric and land surface models. There are various approximations of differ- ent aspects of potential and saturated temperatures to calcu- late θE (Betts and Dugan, 1973; Simpson, 1978); however, many of them have large errors. These errors are compared in Bolton (1980), and Eq. (39) (Bolton’s formulation) is up to an order of magnitude more accurate: where e is the parcel vapor pressure (mb) (using CLM4.5, this is the 2 m vapor pressure), and r is the mixing ratio (g kg−1) (this is converted from the 2 m height Q to r in CLM4.5). Third, the parcel is raised to a great height where all latent heat is transferred to the air parcel, and the water is rained out, giving the solution to θE. There are many methods for representing this process. The analytical solution (Holton, 1972) is computationally prohibitive in atmospheric and land surface models. There are various approximations of differ- ent aspects of potential and saturated temperatures to calcu- late θE (Betts and Dugan, 1973; Simpson, 1978); however, many of them have large errors. These errors are compared in Bolton (1980), and Eq. (39) (Bolton’s formulation) is up to an order of magnitude more accurate: where the combination of equations’ initial guesses is valid from 1050 mb down to 100 mb. J. R. Buzan et al.: Implementation and comparison of a suite of heat stress metrics Our results show that there are two regimes of heat stress – ex- treme moisture and extreme temperature – yet all of the most extreme heat stress events are tied to maximum moisture. Our approach has limitations. None of the metrics in the HumanIndexMod include the effects of solar and thermal www.geosci-model-dev.net/8/151/2015/ Geosci. Model Dev., 8, 151–170, 2015 J. R. Buzan et al.: Implementation and comparison of a suite of heat stress metrics Appendix A: Moist thermodynamics The initial guess for Tw is based upon regions where the second-order derivative of θE reaches a linear relationship with variations in Tw and λ. Two coefﬁcients are derived (Davies-Jones, 2008): Davies-Jones (2008) shows multiple methods of comput- ing Tw, and we implemented the most accurate equations, described below. We introduce terminology to describe the Davies-Jones (2008) calculation. All temperature subscripts that are capitalized are in kelvin, while lower case are in de- grees Celsius. κd is the Poisson constant for dry air (0.2854), and λ is the inverse (3.504). Many of the following equations are scaled using non-dimensional pressure (also known as the Exner function), π: k1 = −38.5π2 + 137.81π −53.737, (A6) k2 = −4.392π2 + 56.831π −0.384. (A7) (A6) (A7) (A6) (A7) The initial guess of Tw for coldest temperatures is Tw = TE −C − Ars (TE,π) 1 + Ars (TE,π) ∂ln(es) ∂TE , (A8) (A8) π = p p0 1/ λ, (A1) (A1) where C is freezing temperature, A is a constant (2675), and rs is the saturated mixing ratio. The evaluation of errors at various pressures necessitated that Davies-Jones develop a regression line on colder regions of the initial guess: where p is the pressure (mb), and p0 is a reference pressure (1000 mb). To deﬁne Tw (the wet bulb temperature), we solve for the equivalent potential temperature, θE. Determining θE is a three-step process. First, we solve for the lifting condensa- tion temperature (TL): C TE λ > D (π);D = 0.1859 p p0 + 0.6512 −1 , (A9) (A9) TL = 1 1 T −55 −ln(RH/ 100) 2840 + 55, (A2) (A2) where D is calculating transition points between quadratic ﬁts to the second-order derivatives of θE. Tw for all other tem- perature regimes is governed by where T is the parcel temperature (kelvin). For example, we use the 2 m air temperature in CLM4.5. RH (%) is taken at the same height as T . TL (Eq. A2), from Eq. (22) in Bolton (1980), is the temperature at which a parcel that is lifted, following a dry adiabatic lapse rate, begins to con- dense. Second, as the air rises further, the parcel now follows a moist potential temperature, θDL: Tw = k1(π) −1.21cold −1.45hot− (k2(π) −1.21cold) C TE λ + 0.58 C TE λ ! www.geosci-model-dev.net/8/151/2015/ Geosci. Model Dev., 8, 151–170, 2015 Appendix A: Moist thermodynamics Following the initial guess, up to two iterations using the Newton–Raphson method are required to reach the true wet bulb temperature. Using TW, saturation vapor pressure is solved by the August–Roche– Magnus formulation of the Clausius–Clayperon equation (Bolton, 1980; Lawrence, 2005): es (TW) = 6.112exp a (TW −C) TW −C + b , (A13) (A13) where es is in mb, and a and b are constants. The saturation mixing ratio, rs, is dependent on es: θE = θDLexp 3.036 TL −0.001788 r (1 + 0.000448r) . (A4) rs (TW) = εes (TW) p0πλ −es (TW) , (A14) (A4) (A14) Equivalent temperature, TE, is θE scaled by π: where ε is a constant (∼0.622). Following Davies-Jones, we use the derivative of the ARM equation for calculating the where ε is a constant (∼0.622). Following Davies-Jones, we use the derivative of the ARM equation for calculating the (A5) TE = θEπ. www.geosci-model-dev.net/8/151/2015/ www.geosci-model-dev.net/8/151/2015/ Geosci. Model Dev., 8, 151–170, 2015 www.geosci-model-dev.net/8/151/2015/ J. R. Buzan et al.: Implementation and comparison of a suite of heat stress metrics 166 J. R. Buzan et al.: Implementation and comparison of a suite of heat stress metrics J. R. Buzan et al.: Implementation and comparison of a suite of heat stress metrics 166 derivative of rs: The derivative of the function Eq. (A18) is required for the Newton Raphson method: derivative of rs: ∂l ( ) b The derivative of the function Eq. (A18) is required for the Newton–Raphson method: The derivative of the function Eq. (A18) is required for the Newton–Raphson method: The derivative of the function Eq. (A18) is required for the Newton–Raphson method: derivative of rs: ∂ln(es) ∂TW = ab (TW −C + b)2 , (A15) ∂es ∂TW = es ∂ln(es) ∂TW , (A16) ∂rs ∂TW π = εp (p −es (TW))2 ∂es ∂TW . (A17) Newton–Raphson method: f ′ (TW;π) = −λ 1 TW + κd (p −es (TW)) ∂es ∂TW + ∂G ∂TW π , (A20) where the derivative of G(TW;π) is (A20) ∂G ∂TW π = −3036 rs (TW) + 0.448r2 s (TW) T 2 W 3036 TW −1.78 (1 + 2(0.448rs (TW))) ∂rs ∂TW π (A21) Now, we return to θE and substitute TWforTL: f (TW;π) = C TW λ 1 − es p0πλ κdλ exp(−λG(TW;π)), (A18) ∂G ∂TW π = −3036 rs (TW) + 0.448rs (TW) T 2 W 3036 TW −1.78 (1 + 2(0.448rs (TW))) ∂rs ∂TW π (A21) f (TW;π) = C TW λ 1 − es p0πλ κdλ exp(−λG(TW;π)), (A18) (A21) (A18) (A18) and, due to the linear relationship of the second-order deriva- tive of Eq. (A18), we may accelerate the Newton–Raphson method using the initially calculated TW and TE: where and, due to the linear relationship of the second order deriva tive of Eq. (A18), we may accelerate the Newton–Raphson h d i h i i i ll l l d T d T where G(TW;π) = 3036 TW −1.78 h rs (TW;π) + 0.448r2 s (TW;π) i . (A19) method using the initially calculated TW and TE: Tw = Tw −f (TW;π) − C TE λ f ′ (TW;π) . (A22) G(TW;π) = 3036 TW −1.78 h π) = TW −1.78 h rs (TW;π) + 0.448r2 s (TW;π) i . Code availability Medicine Natick MA Biophysics and Biomedical Modeling Div, 2005. We will make the HumanIndexMod available at the Univer- sity of New Hampshire Data Discover Center New Hamp- shire Climate section. The NSF-funded New Hampshire EPSCoR Ecosystem and Society Project manage this data archive. Additionally, we will upload the HumanIndexMod to http://Data.gov, a free repository for data, metrics, and re- sults for public use. The United States government manages this repository. Betts, A. K. and Dugan, F. J.: Empirical formula for saturation pseudoadiabats and saturation equivalent potential temperature, J. Appl. Meteorol., 12, 731–732, 1973. pp Bolton, D.: The computation of equivalent potential temperature, Mon. Weather Rev., 108, 1046–1053, 1980. Brake, D. J.: Calculation of the natural (unventilated) wet bulb tem- perature, psychrometric dry bulb temperature and wet bulb globe temperature from standard psychrometric measurements, J. Mine Vent. Soc. S. Afr., 54, 108–112, 2001. Breckenridge, J. R. and Goldman, R. F.: Solar heat load in man, J. Appl. Physiol., 31, 659–663, 1971. Acknowledgements. Computing resources from Information Technology at Purdue University supported this research. J. R. Buzan thanks the reviewers for helpful and constructive comments. J. R. Buzan thanks Aaron Goldner, Jacob Carley, and Nick Herold for helpful comments and support. He also thanks his friends and family for their continued, unwavering, support. K. W. Oleson acknowledges support from the NCAR WCIASP and from NASA grant NNX10AK79G (the SIMMER project). NCAR is sponsored by the National Science Foundation. M. Huber acknowledges support from the NSF-funded New Hampshire EPSCoR Ecosystem and Society Project. Bröde, P., Krüger, E. L., Rossi, F. 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https://openalex.org/W3047542442	https://zenodo.org/records/3957683/files/O0qJFjcZeG.pdf	English	null	Kinematical Analysis of Off-Spin Bowling at Club level Cricket	Global educational studies review	2,019	cc-by	6,313	Assistant Professor, Department of Education & Research, Gomal University, D. I. Khan, KP, Pakistan. Email: malikamiratta@gmail.com †Subject Specialist, Department of Education, Gomal University, D. I. Khan, KP, Pakistan. ‡Lecturer, Department of Education & Research, Gomal University, D. I. Khan, KP, Pakistan. !" $%%&' "#$%&"''( ("$%%&' "#$%&()$% +)' ++,-../01/231245.'$1('#$(.56741"$'89:;&:<1$( ./01 :;= &/1 : 9"$'8< 234(5' "' > ($ !" $%%&' "#$%&"''( ("$%%&' "#$%&()$% +)' ++,-../01/231245.'$1('#$(.56741"$'89:;&:<1$( ./01 :;= &/1 : 9"$'8< 234(5' "' > ($ )"$%%&' "#$%&"''( ,-$' '$1('#$(.56741"$'89:;&:<1$( Key Words: Relationship, Population Configuration, Enrollment, Children, Primary Level Key Words: Relationship, Population Configuration, Enrollment, Children, Primary Level Introduction Socrates defines education as, “Education is the mean that helps in searching the truth. Truth is hidden in the minds of human being and they should be aware of that. Rousseau: Education is the development of human nature in free environment. John Dewey: “education is a process of living through a continuous reconstruction of experiences”. It means that education is the development of the whole personality of an individual. In Pakistan there are four levels of education. Primary education, Secondary education, College and University education. Primary education is the first stage of compulsory education. It is preceded by preschool or nursery education and is followed by secondary education. Relationship of Population Configuration with Enrollment of Children at Primary Level of District Bannu Qayyum Nawaz† Hafiz Azmat Ullah‡ Malik Amer Atta Abstract: The Problem under study was the relationship of population composition with enrollment of children at primary level. The study was significant because we can make forecasting of educational plans keeping in view the relationship. All the Primary School of the District Bannu was included in the population for the study. The sample was 05 Urban Male School, 05 Urban Female School, 05 Rural Male School, and 05 Rural Female School. The research instrument was a questionnaire. Through this questionnaire data was collected regarding schools from 2013 to 2019 in different classes. The null hypothesis that there is no relationship between the population composition and enrollment was tested. The result shows that the correlation between the enrollment and the population growth is 0.98. It is high correlation which indicates the strong relationship between the number of enrollment of student and population Increasing Population and Standard of Life. (it’s Positive & Negative Aspects) Iqbal (2017) Pakistan is a developing country & its whole population is approximately (16,000,0000) sixteen crores. Now the population of Pakistan is increasing very rapidly. If we imagine about increasing population, we feel hesitation but it has some positive aspects too. Which is the following: - The foremost positive aspect of increasing population is that it brings power and strength. About it we have a wise-saying that “majority is authority”. Now in our country those people who are in majority they have well and stable back. But those who are in minority they consider inferior. So, we can say that strength and power can be brought through majority that why increasing population is considered beneficent (Ahmad Maqbool, 2006). Second is also positive aspect of increasing population that the people who are dominant in majority are well acquainted. Everyone knows them well because of their cost and spreading influence. But those who are less not so well known. That’s why we many say that increasing population leads us to a good name and fame (Katozi Ali Murad, 2005). Third, increasing population has a very important aspect because our religion permits us to increase population so that to increase religious color in the world. Now if we go through according to the teachings of Islam we must abolish family planning because it is a negation of our religion and try to increase our population (Iqbal, 2017). Next, the more we increase the more we will progress. If we our *Assistant Professor, Department of Education & Research, Gomal University, D. I. Khan, KP, Pakistan. Email: malikamiratta@gmail.com †Subject Specialist, Department of Education, Gomal University, D. I. Khan, KP, Pakistan. ‡Lecturer, Department of Education & Research, Gomal University, D. I. Khan, KP, Pakistan. Malik Amer Atta, Qayyum Nawaz and Hafiz Azmat Ullah population increases, we will have health facility because more of the people will enter in a health department. Many of the people will cultivate agriculture and bring food prosperity etc. (Tanvir, 2004). According to educational survey of Abadiat (Ministry of Education, 1999) population has negative aspects which are the following: - • First of all, we have our health problem. It is a negative aspect of increasing population. Because in our country there is a lack of physicians and hospitals, if our population increases day by day then we will face difficulty in providing health case to our patients. Increasing Population and Standard of Life. (it’s Positive & Negative Aspects) So, we should adopt a well family planning according to our resources. • Secondly, it is another negative aspect of increasing population which is economical problem. Our country Pakistan is not so stable economically to provide financial to each and every individual whether in ease of jobs or education etc. that’s why our population should be balance to our economy. Otherwise we will face difficulty. • Secondly, it is another negative aspect of increasing population which is economical problem. Our country Pakistan is not so stable economically to provide financial to each and every individual whether in ease of jobs or education etc. that’s why our population should be balance to our economy. Otherwise we will face difficulty. • Another negative impact of increasing population has on our education. It can affect our education because the inhabitants of our country are economically weak. They can’t afford more expenditure because their income is small than their outcome. That’s why if they increase their population, they will be unable to provide a proper education to their children. That’s why I suggest that everyone should go parallel with their expenditure. • Next, increasing population leads us to a lack of confidence. They destroy our determination and leads our children to confusion because in an increasing population. Children have no their childhood. They can’t find a thing for which they demand. So it should be a balanced one because it is the cry and demand of the day. • Next, increasing population leads us to a lack of confidence. They destroy our determination and leads our children to confusion because in an increasing population. Children have no their childhood. They can’t find a thing for which they demand. So it should be a balanced one because it is the cry and demand of the day. Research Objectives o know the relationship between male and female students’ enrollment in primary schoo i) To know the relationship between male and female students’ enrollment in primary school. ii) To know the difference of enrollment between rural and urban primary schools i) To know the relationship between male and female students’ enrollment in primary school. o know the difference of enrollment between rural and urban primary schools. ii) To know the difference of enrollment between rural and urban primary schools. iii) To find out the relationship b/w population composition and enrollment at primary level. iii) To find out the relationship b/w population composition and enrollment at primary level. Problem Statement The problem under study was “to investigate the relationship of population composition with enrollment of children at primary level.” Bad Effects of Population Increase on the Education In Pakistan population growth is, Problem Number one of Pakistan. Higher growth rates of population have very serious effects on various phases of a country. The major reason of higher education rate of developed countries is its decrease in eighteen years old population (Ministry of education 1992). One of the effects of higher population growth rates is on the quality of education. That as a country’s overall development and living standard depends upon education, the higher quality of education leads the nation towards the development and prosperity. But the higher population rates effect the quality of education and the people have to substitute the quality or quantity. Because it becomes more difficult to advice the quality education due to limited resources. Suppose a father who has two children can education them easily and if a father has six children then he will have to face various problems due to which he could not educate well and they become a burden on him. So, we can compare counties population with its available resources. If country population is growing rapidly, obviously it will affect the quality of education (Baker, 2009) Research Sample wing sample was used for the research purpose in District Bannu. Following sample was used for the research purpose in District Bannu. Following sample was used for the research purpose in District Bannu. 1. Urban Male School = 05 2. Urban Female School = 05 3. Rural Male School = 05 4. Rural Female School = 05 Total = 20 Research Instrument g p p p 1. Urban Male School = 05 2. Urban Female School = 05 3. Rural Male School = 05 4. Rural Female School = 05 Total = 20 1. Urban Male School 2. Urban Female School = 05 3. Rural Male School = 05 4. Rural Female School = 05 Total = 20 Research Instrument 2. Urban Female School 3. Rural Male School Significance of the Study This study was significant due to the following reasons. This study was significant due to the following reasons. i) This study tells as about the male and female student’s enrollment in the primary schools of the district Bannu due to which we can easily plane for the education of these children. Also, the i) This study tells as about the male and female student’s enrollment in the primary schools of the district Bannu due to which we can easily plane for the education of these children. Also, the Global Educational Studies Review (GESR) 22 ationship of Population Configuration with Enrollment of Children at Primary Level of District Bannu strength of enrollment helps in the decision-making process for the education for the administration. ii) This study also tells us about the rich and poor students enrollment in the primary schools of the district Bannu, which is very helpful information, because on the behalf of this information we can advertise the education in those areas of the district and in those stratus of the district where there is less motivation of male and female students towards the education. Mostly the poor peoples can’t effort the education for their children, so this study not only highlights such problems but also helps in the education of for flung areas of the district. iii) This study tells us about the education level of rural and urban areas, also about the male and female children. Mostly the people in this district don’t send their female children in school, so this study in a type of effort to motivate such parents towards education. iii) This study tells us about the education level of rural and urban areas, also about the male and female children. Mostly the people in this district don’t send their female children in school, so this study in a type of effort to motivate such parents towards education. Research Instrument The research instrument was a questionnaire. Through this questionnaire data was collected regarding schools from 2013 to 2019 in different classes. Research Methodology Research Population All the Primary School of the District Bannu was included in the population for the study Research Hypotheses H01: there is no significance difference of enrollment between male and female students in primary schools. H02: there is no significance difference of enrollment between rural and urban primary schoo H02: there is no significance difference of enrollment between rural and urban primary schools. H03: there is no relationship between population compositions with enrollment. Limitation There was no such instrument for the collection of record from the primary schools so the researcher developed questionnaires were used for the collection of data. Also the schools can’t maintain the commutative record of each and every student, so only available record was used for the purpose. Delimitation Following was delimitation of the study. Study was delimited to ten male and ten female public schools in district Bannu Research Methodology Research Population Research Procedure Keeping in view the experts views the questionnaire was developed. The researcher personally visited the schools and distributed the questionnaire. The data thus collected in questionnaire was arranged in the form of tables. Then for the purpose of data analysis Co-efficient of correlation was used as a statistical tool. Vol. IV, No. I (2019) Vol. IV, No. I (2019) 23 Malik Amer Atta, Qayyum Nawaz and Hafiz Azmat Ullah 24 Global Educational Studies Review (GESR) Statistical Analysis Co-efficient of correlation was used as statistical technique. Presentation and Analysis of Data Table 1. School GGPS Ghazimarjan (Rural) 1st class Strength in 2013 Strength in 2014 Strength in 2015 Strength in 2016 Strength in 2017 Strength in 2018 Strength in 2019 6 9 11 12 15 17 18 2nd class 5 8 10 13 16 18 20 3rd class 7 10 12 14 17 19 21 4th class 6 8 10 13 15 17 19 5th class 4 7 9 12 14 16 18 Total 28 42 52 64 77 87 96 Table 2. School GGPS Awalzoman (Rural) 1st class Strength in 2013 Strength in 2014 Strength in 2015 Strength in 2016 Strength in 2017 Strength in 2018 Strength in 2019 9 11 14 16 13 15 17 2nd class 8 9 12 14 15 16 18 3rd class 10 12 15 13 14 17 19 4th class 12 14 16 19 15 13 18 5th class 11 13 15 18 20 19 21 Total 50 59 72 80 77 80 93 Table 3. School GGPS Mir Salam Khan (Rural) 1st class Strength in 2013 Strength in 2014 Strength in 2015 Strength in 2016 Strength in 2017 Strength in 2018 Strength in 2019 8 10 13 15 18 21 24 2nd class 10 12 15 17 20 23 25 3rd class 9 11 14 16 18 20 22 4th class 7 10 15 17 19 22 24 5th class 6 9 12 14 17 20 23 Total 40 52 69 79 92 106 118 Table 4. Research Procedure School GGPS Bandar Kala (Rural) 1st class Strength in 2013 Strength in 2014 Strength in 2015 Strength in 2016 Strength in 2017 Strength in 2018 Strength in 2019 5 7 10 12 14 17 19 2nd class 4 6 9 11 13 15 18 3rd class 6 8 10 13 15 17 20 4th class 5 7 9 11 13 15 19 5th class 4 6 8 12 15 17 20 Total 24 34 46 59 70 81 96 Table 5. School GGPS Jandukhel (Rural) 1st class Strength in 2013 Strength in 2014 Strength in 2015 Strength in 2016 Strength in 2017 Strength in 2018 Strength in 2019 6 8 10 13 15 17 19 2nd class 5 7 9 11 13 15 17 3rd class 5 8 11 13 15 18 20 Statistical Analysis Co-efficient of correlation was used as statistical technique. Presentation and Analysis of Data Presentation and Analysis of Data Table 1. School GGPS Ghazimarjan (Rural) Presentation and Analysis of Data Table 1. School GGPS Ghazimarjan (Rural) Table 2. School GGPS Awalzoman (Rural) Table 3. School GGPS Mir Salam Khan (Rural) Table 4. School GGPS Bandar Kala (Rural) Table 5. School GGPS Jandukhel (Rural) Global Educational Studies Review (GESR) 24 24 Relationship of Population Configuration with Enrollment of Children at Primary Level of District Bannu 4th class 4 7 9 12 14 16 18 5th class 5 8 10 13 15 17 19 Total 25 38 49 62 72 83 93 Table 6. School GPS Gul Bazar (Rural) 1st class Strength in 2013 Strength in 2014 Strength in 2015 Strength in 2016 Strength in 2017 Strength in 2018 Strength in 2019 13 19 27 33 47 49 58 2nd class 15 20 26 31 38 45 60 3rd class 13 18 25 30 37 40 45 4th class 16 21 27 32 42 50 55 5th class 20 28 35 43 47 51 56 Total 77 106 140 169 205 235 274 Table 7. Research Procedure School GPS Ayaz Khan (Rural) 1st class Strength in 2013 Strength in 2014 Strength in 2015 Strength in 2016 Strength in 2017 Strength in 2018 Strength in 2019 15 20 24 31 37 43 50 2nd class 17 21 26 30 38 44 52 3rd class 20 25 29 36 41 37 43 4th class 18 21 25 32 37 42 49 5th class 14 18 23 26 30 36 42 Total 84 105 127 155 183 202 236 Table 8. School GPS Alamdin (Rural) 1st class Strength in 2013 Strength in 2014 Strength in 2015 Strength in 2016 Strength in 2017 Strength in 2018 Strength in 2019 11 20 27 33 40 48 55 2nd class 7 13 20 27 35 40 47 3rd class 10 16 22 30 37 44 52 4th class 12 20 26 21 19 23 31 5th class 15 18 16 13 11 9 7 Total 55 87 111 124 142 164 192 Table 9. School GPS Laotykala (Rural) 1st class Strength in 2013 Strength in 2014 Strength in 2015 Strength in 2016 Strength in 2017 Strength in 2018 Strength in 2019 16 20 24 27 30 28 33 2nd class 19 22 25 28 32 35 38 3rd class 21 24 27 30 33 30 35 4th class 23 26 30 34 37 40 43 5th class 25 28 32 35 40 38 41 Total 104 120 138 154 172 171 190 Table 10. School GPS Azim Kala (Rural) 1st class Strength in 2013 Strength in 2014 Strength in 2015 Strength in 2016 Strength in 2017 Strength in 2018 Strength in 2019 19 24 29 34 39 44 49 2nd class 16 19 23 28 33 36 42 3rd class 14 20 24 27 31 35 40 Relationship of Population Configuration with Enrollment of Children at Primary Level of District Bannu ationship of Population Configuration with Enrollment of Children at Primary Level of District Bannu Table 7. Research Procedure School GPS Ayaz Khan (Rural) 1st class Strength in 2013 Strength in 2014 Strength in 2015 Strength in 2016 Strength in 2017 Strength in 2018 Strength in 2019 15 20 24 31 37 43 50 2nd class 17 21 26 30 38 44 52 3rd class 20 25 29 36 41 37 43 4th class 18 21 25 32 37 42 49 5th class 14 18 23 26 30 36 42 Total 84 105 127 155 183 202 236 Table 8. School GPS Alamdin (Rural) 1st class Strength in 2013 Strength in 2014 Strength in 2015 Strength in 2016 Strength in 2017 Strength in 2018 Strength in 2019 11 20 27 33 40 48 55 2nd class 7 13 20 27 35 40 47 3rd class 10 16 22 30 37 44 52 4th class 12 20 26 21 19 23 31 5th class 15 18 16 13 11 9 7 Total 55 87 111 124 142 164 192 Table 9. School GPS Laotykala (Rural) 1st class Strength in 2013 Strength in 2014 Strength in 2015 Strength in 2016 Strength in 2017 Strength in 2018 Strength in 2019 16 20 24 27 30 28 33 2nd class 19 22 25 28 32 35 38 3rd class 21 24 27 30 33 30 35 4th class 23 26 30 34 37 40 43 th Table 7. School GPS Ayaz Khan (Rural) Table 8. School GPS Alamdin (Rural) Table 9. School GPS Laotykala (Rural) Table 10. School GPS Azim Kala (Rural) 25 Malik Amer Atta, Qayyum Nawaz and Hafiz Azmat Ullah 4th class 17 21 25 29 27 32 36 5th class 18 22 26 24 28 31 35 Total 84 106 127 142 158 178 202 Table 11. School GGPS NO 1 City (Urban) 1st class Strength in 2013 Strength in 2014 Strength in 2015 Strength in 2016 Strength in 2017 Strength in 2018 Strength in 2019 13 16 18 21 24 27 30 2nd class 11 14 16 17 15 20 22 3rd class 13 15 17 21 24 27 31 4th class 15 17 19 23 26 29 32 5th class 14 16 20 22 25 30 34 Total 66 78 90 104 114 133 149 Table 12. Research Procedure School GGPS NO 2 City (Urban) 1st class Strength in 2013 Strength in 2014 Strength in 2015 Strength in 2016 Strength in 2017 Strength in 2018 Strength in 2019 12 14 17 19 21 23 26 2nd class 11 13 16 18 20 22 25 3rd class 10 12 15 17 19 21 24 4th class 9 11 14 16 18 22 25 5th class 8 11 13 17 19 23 24 Total 50 61 75 87 97 111 124 Table 13. School GGPS NO 3 City (Urban) 1st class Strength in 2013 Strength in 2014 Strength in 2015 Strength in 2016 Strength in 2017 Strength in 2018 Strength in 2019 13 15 17 20 23 25 27 2nd class 12 14 16 19 22 24 26 3rd class 10 13 15 17 20 22 25 4th class 9 12 14 16 19 21 24 5th class 8 11 13 15 17 19 23 Total 52 65 75 85 101 111 125 Table 14. School GGPS NO 4 City (Urban) 1st class Strength in 2013 Strength in 2014 Strength in 2015 Strength in 2016 Strength in 2017 Strength in 2018 Strength in 2019 14 16 19 22 25 28 32 2nd class 12 14 17 20 23 25 27 3rd class 11 13 15 18 20 23 26 4th class 9 12 14 17 19 22 25 5th class 8 10 13 16 18 20 24 Total 54 65 78 93 105 118 134 Table 15. School GGPS Mandon (Urban) 1st class Strength in 2013 Strength in 2014 Strength in 2015 Strength in 2016 Strength in 2017 Strength in 2018 Strength in 2019 15 17 20 22 25 27 30 2nd class 14 16 17 20 22 25 27 3rd class 11 14 16 18 20 22 25 Malik Amer Atta, Qayyum Nawaz and Hafiz Azmat Ullah Malik Amer Atta, Qayyum Nawaz and Hafiz Azmat Ullah Malik Amer Atta, Qayyum Nawaz and Hafiz Azmat Ullah Table 12. Research Procedure School GGPS NO 2 City (Urban) 1st class Strength in 2013 Strength in 2014 Strength in 2015 Strength in 2016 Strength in 2017 Strength in 2018 Strength in 2019 12 14 17 19 21 23 26 2nd class 11 13 16 18 20 22 25 3rd class 10 12 15 17 19 21 24 4th class 9 11 14 16 18 22 25 5th class 8 11 13 17 19 23 24 Total 50 61 75 87 97 111 124 Table 13. School GGPS NO 3 City (Urban) 1st class Strength in 2013 Strength in 2014 Strength in 2015 Strength in 2016 Strength in 2017 Strength in 2018 Strength in 2019 13 15 17 20 23 25 27 2nd class 12 14 16 19 22 24 26 3rd class 10 13 15 17 20 22 25 4th class 9 12 14 16 19 21 24 5th class 8 11 13 15 17 19 23 Total 52 65 75 85 101 111 125 Table 14. School GGPS NO 4 City (Urban) 1st class Strength in 2013 Strength in 2014 Strength in 2015 Strength in 2016 Strength in 2017 Strength in 2018 Strength in 2019 14 16 19 22 25 28 32 2nd class 12 14 17 20 23 25 27 3rd class 11 13 15 18 20 23 26 4th class 9 12 14 17 19 22 25 5th class 8 10 13 16 18 20 24 Total 54 65 78 93 105 118 134 Table 12. School GGPS NO 2 City (Urban) Table 13. School GGPS NO 3 City (Urban) Table 14. School GGPS NO 4 City (Urban) Table 15. School GGPS Mandon (Urban) Global Educational Studies Review (GESR) 26 Relationship of Population Configuration with Enrollment of Children at Primary Level of District Bannu 4th class 9 12 15 17 19 21 24 5th class 7 10 12 15 17 20 23 Total 56 69 80 92 103 115 129 Table 16. School GPS NO 1 City (Urban) 1st class Strength in 2013 Strength in 2014 Strength in 2015 Strength in 2016 Strength in 2017 Strength in 2018 Strength in 2019 20 26 32 37 42 50 60 2nd class 22 28 34 40 45 51 62 3rd class 19 25 30 36 41 48 55 4th class 24 29 34 43 50 56 63 5th class 106 133 162 192 219 254 296 Table 17. Research Procedure School GPS NO 2 City (Urban) 1st class Strength in 2013 Strength in 2014 Strength in 2015 Strength in 2016 Strength in 2017 Strength in 2018 Strength in 2019 19 24 29 34 40 45 52 2nd class 18 23 28 33 39 44 50 3rd class 16 20 25 30 35 41 49 4th class 15 19 26 31 37 43 51 5th class 13 18 24 29 34 40 45 Total 81 104 132 157 185 213 247 Table 18. School GPS NO 3 Cityn(Urban) 1st class Strength in 2013 Strength in 2014 Strength in 2015 Strength in 2016 Strength in 2017 Strength in 2018 Strength in 2019 20 24 27 31 36 40 46 2nd class 19 22 26 30 35 39 43 3rd class 17 20 25 29 34 38 41 4th class 15 18 23 26 29 `34 40 5th class 13 17 21 25 30 33 38 Total 84 101 122 141 164 184 208 Table 19. School GPS Tehsil Street City (Urban) 1st class Strength in 2013 Strength in 2014 Strength in 2015 Strength in 2016 Strength in 2017 Strength in 2018 Strength in 2019 18 22 25 29 33 36 41 2nd class 16 20 24 30 34 37 40 3rd class 15 19 23 29 38 45 50 4th class 19 25 31 38 43 48 51 5th class 21 26 30 36 42 46 50 Total 86 112 133 162 190 212 232 Table 20. School GPS Sorani (Urban) 1st class Strength in 2013 Strength in 2014 Strength in 2015 Strength in 2016 Strength in 2017 Strength in 2018 Strength in 2019 20 21 26 32 38 44 51 2nd class 19 23 28 33 39 45 52 3rd class 18 22 29 35 40 43 49 Relationship of Population Configuration with Enrollment of Children at Primary Level of District Bannu Table 17. School GPS NO 2 City (Urban) 1st class Strength in 2013 Strength in 2014 Strength in 2015 Strength in 2016 Strength in 2017 Strength in 2018 Strength in 2019 19 24 29 34 40 45 52 2nd class 18 23 28 33 39 44 50 3rd class 16 20 25 30 35 41 49 4th class 15 19 26 31 37 43 51 5th class 13 18 24 29 34 40 45 Total 81 104 132 157 185 213 247 Table 18. Research Procedure Variable n Total Sum of square of total Correlation No of students 7 14744 43830758 0.98 Population 7 1056000000000 1596958000000000000000000000 Table 23. Showing the correlation between the number of students and population growth from 2013 to 2019 Year No of student Population 2013 1298 141500000 2014 1644 142700000 2015 2014 148000000 2016 2360 152000000 2017 2687 154000000 2018 3058 158000000 2019 3483 159800000 g the correlation between the number of students and population growth from 2013 to Correlation between the Enrollment and the Population Result: It is high correlation. It shows the strong relationship between the number of enrollments of student and population growth. Variable n Total Sum of square of total Correlation No of students 7 14744 43830758 0.98 Population 7 1056000000000 1596958000000000000000000000 Correlation between the Enrollment and the Population Research Procedure School GPS NO 3 Cityn(Urban) 1st class Strength in 2013 Strength in 2014 Strength in 2015 Strength in 2016 Strength in 2017 Strength in 2018 Strength in 2019 20 24 27 31 36 40 46 2nd class 19 22 26 30 35 39 43 3rd class 17 20 25 29 34 38 41 4th class 15 18 23 26 29 `34 40 5th class 13 17 21 25 30 33 38 Total 84 101 122 141 164 184 208 Table 19. School GPS Tehsil Street City (Urban) 1st class Strength in 2013 Strength in 2014 Strength in 2015 Strength in 2016 Strength in 2017 Strength in 2018 Strength in 2019 18 22 25 29 33 36 41 2nd class 16 20 24 30 34 37 40 3rd class 15 19 23 29 38 45 50 4th class 19 25 31 38 43 48 51 5th class 21 26 30 36 42 46 50 Total 86 112 133 162 190 212 232 Table 17. School GPS NO 2 City (Urban) Table 17. School GPS NO 2 City (Urban) 1st class Strength in 2013 Strength in 2014 Strength in 2015 Strength in 2016 Strength in 2017 Strength in 2018 Strength in 2019 19 24 29 34 40 45 52 2nd class 18 23 28 33 39 44 50 3rd class 16 20 25 30 35 41 49 4th class 15 19 26 31 37 43 51 5th class 13 18 24 29 34 40 45 Total 81 104 132 157 185 213 247 Table 18. School GPS NO 3 Cityn(Urban) Table 18. School GPS NO 3 Cityn(Urban) 1st class Strength in 2013 Strength in 2014 Strength in 2015 Strength in 2016 Strength in 2017 Strength in 2018 Strength in 2019 20 24 27 31 36 40 46 2nd class 19 22 26 30 35 39 43 3rd class 17 20 25 29 34 38 41 4th class 15 18 23 26 29 `34 40 5th class 13 17 21 25 30 33 38 Total 84 101 122 141 164 184 208 Table 19 School GPS Tehsil Street City (Urban) Table 19. Research Procedure School GPS Tehsil Street City (Urban) 27 Malik Amer Atta, Qayyum Nawaz and Hafiz Azmat Ullah Malik Amer Atta, Qayyum Nawaz and Hafiz Azmat Ullah Malik Amer Atta, Qayyum Nawaz and Hafiz Azmat Ullah 4th class 17 21 28 36 43 48 50 5th class 15 20 26 31 36 40 45 Total 89 107 137 167 196 220 247 Tabale 21. Population in Pakistan Year 2013 2014 2015 2016 2017 2018 2019 Population 14,1500000 14,2700000 148000000 152000000 154000000 158000000 159800000 Table 22. Showing the total enrollment of the student form class 1st to class 5th in the year 2013 to 2019 1st class Strength in 2013 Strength in 2014 Strength in 2015 Strength in 2016 Strength in 2017 Strength in 2018 Strength in 2019 272 343 419 483 569 644 737 2nd class 260 324 397 470 547 619 711 3rd class 255 325 399 472 514 607 692 4th class 261 331 411 483 541 614 699 5th class 250 321 388 452 516 574 644 Total 1298 1644 2014 2360 2687 3058 3483 Table 23. Showing the correlation between the number of students and population growth from 2013 to 2019 Year No of student Population 2013 1298 141500000 2014 1644 142700000 2015 2014 148000000 2016 2360 152000000 2017 2687 154000000 2018 3058 158000000 2019 3483 159800000 Correlation between the Enrollment and the Population Result: It is high correlation. It shows the strong relationship between the number of enrollments of student and population growth. Research Findings Following were the research findings Variable n Total Sum of square of total Correlation No of students 7 14744 43830758 0.98 Population 7 1056000000000 1596958000000000000000000000 wing the total enrollment of the student form class 1st to class 5th in the year 2013 to 2019 Table 22. Showing the total enrollment of the student form class 1st to class 5th in the y Table 23. Showing the correlation between the number of students and population growth from 2013 to 2019 Year No of student Population 2013 1298 141500000 2014 1644 142700000 2015 2014 148000000 2016 2360 152000000 2017 2687 154000000 2018 3058 158000000 2019 3483 159800000 Correlation between the Enrollment and the Population Result: It is high correlation. It shows the strong relationship between the number of enrollments of student and population growth. Research Findings Following were the research findings Following were the research findings • First class enrollment during the years from 2013 to 2019 was, 8 6 6 d ti l • First class enrollment during the years from 2013 to 2019 was 272, 343, 419, 483, 569, 644, and 737 respectively. 272, 343, 419, 483, 569, 644, and 737 respectively. • Second class enrollment during the years from 2013 to 2019 was, 260, 324, 397, 470, 547, 619 and 711 respectively. 260, 324, 397, 470, 547, 619 and 711 respectively. • Third class enrollment during the years from 2013 to 2019 was, g y 255, 325, 399, 472, 514, 607 and 692 respectively. 255, 325, 399, 472, 514, 607 and 692 respectively. • Fourth class enrollment during the years from 2013 to 2019 was, 261, 331, 411, 483, 541, 614 and 699 respectively. • Fifth class enrollment during the years from 2013 to 2019 was, 250, 321, 388, 452, 516, 574 and 644 respectively. • The total enrollment of the year 2013 was 1298 while the population of the year wa Global Educational Studies Review (GESR) Global Educational Studies Review (GESR) 28 Relationship of Population Configuration with Enrollment of Children at Primary Level of District Ban • The total enrollment of the year 2014 was 1644 while the population of the year was 142700000. • The total enrollment of the year 2015 was 2014 while the population of the year was 148000000. • The total enrollment of the year 2016 was 2687 while the population of the year was 154000000. • The total enrollment of the year 2017 was 2687 while the population of the year was 154000000. • The total enrollment of the year 2018 was 3058 while the population of the year was 158000000. • The total enrollment of the year 2019 was 3483 while the population of the year was 159800000 • The total enrollment of the year 2016 was 2687 while the population of the year was 154000000. • The total enrollment of the year 2017 was 2687 while the population of the year was 154000000. • The total enrollment of the year 2018 was 3058 while the population of the year was 158000000. • The total enrollment of the year 2019 was 3483 while the population of the year was 159800000 Research Conclusions From the above findings it can be concluded that: From the above findings it can be concluded that: • The enrollment rate increases with the population growth. • The correlation between the enrollment and the population growth is 0.98. • It is high correlation it shows the strong relationship between the number of enrollments of student and population • It is high correlation it shows the strong relationship between the number of enrollments of student and population Recommendations In the light of the findings of the study following recommendations are In the light of the findings of the study following recommendations are made. • New schools should be established with the increase in population. • Facilities should be increased with the passage of time. • Infrastructure should be increased with the increasing population. • New rooms should be built in the existing schools. • Schools should be upgraded with the increasing population. Vol. IV, No. I (2019) Vol. IV, No. I (2019) Vol. IV, No. I (2019) 29 Malik Amer Atta, Qayyum Nawaz and Hafiz Azmat Ullah Global Educational Studies Review (GESR) Malik Amer Atta, Qayyum Nawaz and Hafiz Azmat Ullah References qbool (2006) Illmi concise dictionary of education, Illmi Kitab Khana, Lahore, Pakistan Baker D, Collins J, Leon J. (2009) Prospects: Quarterly Review of Comparative Education. 4. Vol. 38. Paris: UNESCO; Risk factor or social vaccine? The historical progression of the role of education in HIV/AIDS infection in sub-Saharan Africa; pp. 467–486. Baker D, Collins J, Leon J. (2009) Prospects: Quarterly Review of Comparative Education. 4. Vol. 38. Paris: UNESCO; Risk factor or social vaccine? The historical progression of the role of education in HIV/AIDS infection in sub-Saharan Africa; pp. 467–486. Iqbal. M (2017) Education in Pakistan. Second Edition (Enlarged and revised in 1977), Aziz Publishers, Urdu Bazar Lahore, Pakistan, pp 258. Iqbal. M (2017) Education in Pakistan. Second Edition (Enlarged and revised in 1977), Aziz Publishers, Urdu Bazar Lahore, Pakistan, pp 258. Katozi Ali Murad (2005) Introduction to education, a comprehensive study of education for perspective headmaster and headmistress, Muhammad Sohabib Gul and Shamshad Gul, Peshawar, pp 2-3 Katozi Ali Murad (2005) Introduction to education, a comprehensive study of education for perspective headmaster and headmistress, Muhammad Sohabib Gul and Shamshad Gul, Peshawar, pp 2-3 Ministry of Education (1999) Professional Training". http://www.pbs.gov.pk/content/census-private- educational-institutions-pakistan-1999-2000 Katozi Ali Murad (2005) Introduction to education, a comprehensive study of education for perspective headmaster and headmistress, Muhammad Sohabib Gul and Shamshad Gul, Peshawar, pp 2-3 pp 3 Ministry of Education (1999) Professional Training". http://www.pbs.gov.pk/content/census-private- educational-institutions-pakistan-1999-2000. pp 3 Ministry of Education (1999) Professional Training". http://www.pbs.gov.pk/content/census-private- educational-institutions-pakistan-1999-2000. p 999 Teacher Guide Taleem-e-Abadiat (1999) Ministry of Education with the Collaboration of UNO Fund for UNFPA and UNESCO. Curriculum Bureau, Islamabad. Teacher Guide Taleem-e-Abadiat (1999) Ministry of Education with the Collaboration of UNO Fund for UNFPA and UNESCO. Curriculum Bureau, Islamabad. Teacher Guide Taleem-e-Abadiat (1999) Ministry of Education w UNFPA and UNESCO. Curriculum Bureau, Islamabad. Zaman, T., U. (2004) Study Guide on Higher Education, code 828, Faculty of Education, Teacher Education Department, Allama Iqbal Open University, Islamabad, pp 40. Zaman, T., U. (2004) Study Guide on Higher Education, code 828, Faculty of Education, Teacher Education Department, Allama Iqbal Open University, Islamabad, pp 40. Global Educational Studies Review (GESR) 30
https://openalex.org/W4384297563	https://journal.uinjkt.ac.id/index.php/arabiyat/article/download/28956/pdf	English	null	Students’ Arabic Prokem in the Modern Islamic School Environment	Arabiyat : Jurnal Kebahasaaraban dan Pendidikan Bahasa Arab/Arabiyat: Jurnal Pendidikan Bahasa Arab dan Kebahasaaraban	2,023	cc-by-sa	5,662	Arabiyat : Jurnal Pendidikan Bahasa Arab dan Kebahasaaraban Vol. 10 No. 1, June 2023, 121-135 P-ISSN: 2356-153X; E-ISSN: 2442-9473 doi: http://dx.doi.org/10.15408/a.v10i1.28956 Arabiyat : Jurnal Pendidikan Bahasa Arab dan Kebahasaaraban Vol. 10 No. 1, June 2023, 121-135 P-ISSN: 2356-153X; E-ISSN: 2442-9473 doi: http://dx.doi.org/10.15408/a.v10i1.28956 Abstract The phenomena of using Arabic language in modern Islamic boarding schools can be various since Individuals in contemporary Islamic schools tend to communicate in Arabic for formal and informal occasions, like in Gontor, East Java, a modern Islamic boarding school. The variations of using Arabic language in Gontor can be called Arabic prokem. This problem results from the interaction of speakers from various backgrounds and environments. At the same time, those circumstances make the Arabic prokem in Gontor unique for its dialect. Therefore, library research is applied in this qualitative study using qualitative descriptive as the method. The data were collected from Bahasa Arab Khas Gontor, a book by Hisyam Zaini. This book clearly describes words, sentences, forms, and expressions spoken out by students in Gontor. The result of the study shows that the Arabic prokem used by students in Gontor comes from Indonesian vocabulary, the Arabic prokem is delivered in the form of interjection, the Arabic prokem has the changes of phoneme, and the Arabic prokem has Dhamîr pattern. Introduction As social beings, language is fundamental for humans to communicate. Language has been used as a form of communication since the beginning of human existence (Wicaksono, 2016). Without language, groups of society will not form. Language is one of the most distinctive and human traits that separates humans from other species. The use of language as a means of communication is an integral component of culture (Devianty, 2017). We can understand what is expected of the material and metaphysical universe by using a language with a distinctive linguistic structure (Noermanzah, 2019: 306). Therefore, language reveals a community's cultural identity, which is then known as language or speech community. Experts sometimes use this term to describe majority language users (Baker & Jones, 1998: 96). Language Arabiyât fluctuates due to the variety of social interactions in the community, which is made up of heterogeneous or different populations (Khoirurrohman & Abdan, 2020). In fact, a language community can be formed when a minimum of two people communicate in the same aim with their own characteristics. In addition, a language is a powerful tool that can help a country achieve its national and international goals (Nasution, 2007). Generally, language characteristics spoken out by each speaker are various. Specifically, these characteristics are associated with vocal tract system, gesture of speakers, source of excitation, also segmental and supra-segmental elements in producing language (Rao et al., 2015: 9). These characteristics are influenced by social environment. It produces the principle of language (Ross, 1994: 176). The variety of languages in terms of the speakers consists of a variety of prokem or slang languages. According to Alwasilah (in Rosalina et al., 2020), slang is a form of language characterized by novel and fast changing vocabulary used by a group of individuals, typically young people, to communicate. Young people primarily utilize slang when they dislike the rigid restrictions of their own language. Slang or prokem is a type of informal language spoken within a group to keep information private and set the group apart from others (Fauziah & Suhartono, 2021: 153). The prokem variety is not commonly used by speakers, so its meaning is only understood by minority. Thus, this language tends to be marginalized (Batibo, 2005: 51). However, the marginality does not only mean to be underrepresented because this language intentionally has secret meaning. Arabiyât Prokem consists of various types and forms. Swandy (Swandy, 2017: 4) states that prokem as a kind of slang, formed by reversing the words order, giving insertions, taking acronyms and abbreviations, changing the vowels and the consonants, changing the overall sound or total sound. As the effect, it is difficult to predict the form of prokem since the word change is unsystematic. For some people, the prokem can be changed into strange, funny and unpredicted words (Swandy, 2017: 4). Prokem is classified as non-formal language. In general context, formal and informal languages are in the same position (Marcus, 2007: 15). On the other hand, the position of these languages is different in the context of language politics. There are clear differences when language is used in daily life and in a national event. Prokem is obviously categorized as informal language. In other words, slang may be different with its original words due to the word changes. The slang can be funny, weird, and unpredictable in form and meaning (Swandy, 2017). Slang internal variations are typically transient and slang usage varies over time according to the times (Margiyanti & Yuliayanto, 2021: 165). In Indonesia, there are numerous communities have prokem to communicate. In Jakarta, for example, young people tend to say “elu-elu gua-gua”, “Boljung dah” or “ampe ngacirr”. This prokem is commonly used by modern Betawi people (Susanto, 2008: 28). There is also prokem in Yogyakarta. The prokem here is formed through affixation. It changes the form of original words. For instance, motor in prokem words is dogosku, not dogosnyu (Kurniawati, 2013: 107). In the 1980s, there were numerous regional variances in the slang used by various community groups. It indicates that a certain location has its own distinctive slang (Suhardianto & Suryani, 2018: 164). Gunawan (as cited in Setiawan, 2019:139) stated a new slang language named Alay arose in the 2000s. The Alay language evolved after the widespread use of cell phones with short messaging service (SMS) capabilities. Many previous studies related to slang on social media (Aryanti et al., 2019; Goziyah & Yusuf, 2019; Ismawati, 2020; Istiqomah et al., 2018; Putra & Hartanto, 2020), Indonesian language learning (Juanda, 2012), and novel (Fahrudin & Kharisma, 2021; Jannah et al., 2020). Meanwhile, in this study, the authors focused on students’ Arabic prokem in the Gontor modern Islamic boarding school environment. Prokem is also used in modern Islamic boarding school like Gontor. Introduction A group of people use this language for their community only, not to be known by others. Basically, minority language is only used by certain people. For instance, particular language employed in academic environment. In the field of law, many language terms in law are used by lawyers and judges. These terms must not be familiar for each person. In the field of education, the term may relate to the context of learning and research (August & Hakuta, 1998: 89). Similar to minority languages, prokem is used by specific individuals in particular places. Generally, prokem is far away from standard vocabulary and language structures. Consequently, not all of people know the prokem words and their meaning. Prokem can be found in several places, such as in the night streets where community of transgender, homosexual, drug addicts, criminals or pickpockets gathered (Jatmika, 2009: 31). As explained by Chaer and Agusina, prokem is also known as slang, a language with specific meaning. The meaning is only understood by a community and purposely secreted from other societies. Slang has nothing to do with grammar and phonology, it relates to vocabulary and phonology. Prokem is temporary and undisclosed, so people assume it is negative and bad communities use it. In fact, it is also used for noble reasons. Besides, its undisclosed meaning makes prokem changes often in both the form and the meaning (Abdul & Leonie, 2014). The area's experiences or situations influence the emergence of slang in the young circle. Additionally, the social group, age, and even educational status of the community have an impact on how slang develops among its members (Setiawan, 2018: 215). People should think about how they use slang in social situations because not everyone can understand it (Ulandari, 2018: 2). ARABIYAT, ISSN: 2356-153X, E-ISSN: 2442-9473 122 Method This study is qualitative and the data are classified into primary and secondary. The primary data words or language facts in a book, entitled Bahasa Arab Gontor by Zaini (2013). This book describes words, sentences, structures, and expression spoken in detail. This language fact is main focus in this study. After that, the data are collected, analyzed, interpreted and summarized (Sugiarto, 2017: 8–9). This study's secondary data are journals, articles, and books related to prokem. This study is library research, meaning that the data source associate with library in finding topics in this study (Djiwandono, 2015: 27). The method applied in this study is descriptive qualitative which indicates that focusing the problem in the current condition by describing it, it is not to find causes-effect of the problem (Yusuf, 2016: 63). Through this method, the problem found on language fact is revealed. In short, this study describes real condition of language fact and offers a way to solve it (Kimbal, 2015: 64). Arabiyât For this reason, prokem in Gontor is mostly found in Arabic language. This Arabic prokem is known as Arab ‘Amiyyah. Generally, Arabic language is divided into Arab Fushhâ (formal) and ‘Amiyyah (informal). The Arab Fushhâ can be found in Al- Qur’an and in a formal condition, such as in schools, mosques, or meetings. This Arabic is based on nahwu and sharaf. In contrast, Arab ‘Amiyyah has nothing to do with nahwu and sharaf. Thus, it is easier to learn since this Arabic is commonly used to communicate in daily life (Darmin, 2019: 4). The condition above exposes that Arab Fushhâ is essential to education system in Islamic modern school. Even though there is a fact that this Arabic is influenced by students’ local dialect (Wekke, 2019: 25). There are some factors that form Arabic prokem in Gontor. First, students are required to speak in Arabic. Second, Arabic is used in the classroom and social activity, such as in boarding houses, dining rooms, canteens. Third, students’ Arabic is influenced by dialect of their native language, both on Indonesian and local languages. That what makes Arabic prokem in Gontor is interesting to study. Arabiyât This school is one of pioneers modernization in Islamic boarding school. The change of Gontor from salafi to modern makes Gontor a place to get Islamic education for urban people unfamiliar with conventional and traditional education systems. The characteristic in Gontor is the tight way of disciplinary system and the way of using Arabic and English in daily activities (Kompri, 2018: 22–23). In language learning, Gontor has their own curriculum, which is different with other Islamic modern schools. For example, students in other schools learn Arabic, followed by Nahwu and Sharf and another Islamic knowledge. In Gontor, students are required to speak Arabic in daily life, so that they can master it fast and then learn Nahwu, Sharaf and other Islamic lessons through Arabic books (Zaini, 2013). ARABIYAT, ISSN: 2356-153X, E-ISSN: 2442-9473 123 Result and Discussion Results should be clear and concise. The results should summarize (scientific) findings rather than providing data in great detail. Please highlight differences between your results or findings and the previous publications by other researchers. The writer found interesting Arabic prokem which is not common in general Arabic. This prokem is collected from Bahasa Arab Gontor, a book written by Hisyam Zaimi. The meaning of this prokem cannot be understood if there is no research on it. Actually, this prokem is produced and reproduced by students in Gontor so that they naturally and spontaneously use this prokem in daily life. This also happens because the students must communicate in Arabic. However, since they have their own dialect from their native local language, their Arabic sounds different with the general one. It turns to Arabic prokem in Gontor. The Arabic prokem in Bahasa Arab Gontor by Hisyam Zaini can be seen in the table below. ARABIYAT, ISSN: 2356-153X, E-ISSN: 2442-9473 124 Arabiyât Arabiyât Table 2 Arabic Prokem in Context of Indonesian Languag No Arabic Prokem Be read Meaning 1 قدم دم Qudamdam ancient people 2 يتفنكفنك Yatafangfank funky style 3 جيل Jel Man 4 غندوق Ghanduq Angry 5 كمان Kamaan More 6 كجكز Kajakaz Likewise Table 2 Arabic Prokem in Context of Indonesian Language In the table above, “qudamdam” is Arabic prokem used by students in Gontor which is not found in Arabic language for Arab ‘Amiyyah and Fushhâ. This prokem comes from “qadim” which means ancient or old. “Qudamdam”as prokem gets syllable “dam”. This syllable comes from Indonesian language to show plural thing. Then, the prokem “Yatafankfank” is not rooted from Indonesian but English, “funky”. This Arabic prokem has “funky” as its basic morpheme. In Arabic, funky is similar with “jabaan” or “namtun”. The prokem of “Yatafankfank” is the combination of the third person pronoun "yes mudhâra'ah" and the word "fung" is repeated, so that it becomes "yatafangfank". This prokem refers to his/her funky style. His or her is to show the third person. Next, the prokem “jel” and “ghanduq” are arbitrary. This prokem is naturally formed and students in Gontor familiar with it because it is easy to remember. Then, the prokem “man” comes from English which is similar with “al-rajul” snf not “jel” in Arabic. The prokem “ghanduk” which means angry is similar with “غضة” in Arabic. The use of Arabic prokem in Indonesian language is stated in the table below. Arabiyât Arabiyât Table 1 Prokem Bahasa Arab Gontor Table 1 Table 1 Prokem Bahasa Arab Gontor No Page Arabic Prokem Be read Meaning 1 19 قدم دم Qudamdam ancient people 2 22 يتفنكفنك Yatafankfank funky style 3 25 صاع Sha’ Ostentatious 4 27 هيه Heh Interjection 5 28 طريق- ريق ط Thariq-thariq Walking around 6 35 كجك Kajaka likewise هجا Haja This أفا Affan apology 7 42 مديك Mudik Funny جيل Jel Man 8 47 خذت Khudztin take كمطرا Kematharan Get rained on 9 48 باص Bashi Fusty غندوق Ghanduq Angry 10 67 كمان Kaman More 11 73 هذاؤه Hadzauh This ذلكه Dzalikuh That 12 82 إنسانه Insanuh The person إدّمه Iddamuh The dish 13 90 ط فق اله Faqath lah Just 14 119 ال ماذا ماذا Laa madza madza Never mind Prokem Bahasa Arab Gontor Based on the data above, Arabic prokem used by students in Gontor can be divided into several types: 1. Arabic Prokem in Context of Indonesian Language (Indonesian Arabic) This kind of prokem is rooted from Arabic by adding Indonesian syllables, as seen in this following table. ARABIYAT, ISSN: 2356-153X, E-ISSN: 2442-9473 125 Arabiyât Arabiyât Table 5 Examples of Arabic Prokem from Indonesian Language Examples of Arabic Prokem from Indonesian Language Examples of Arabic Prokem from Indonesian Language Meaning in Indonesian Language Arabic Prokem Page Making laugh صناع مديك 44 You’ve got me angry صناع غندوق فقط أنت ذلك 85 Examples of Arabic Prokem from Indonesian Language Meaning in Indonesian Language Arabic Prokem Page Making laugh صناع مديك 44 You’ve got me angry صناع غندوق فقط أنت ذلك 85 2. Arabic Prokem in Indonesian Vocabulary A b k d f d Arabic prokem rooted from Indonesian language is created but using Indonesian vocabulary, but it is pronounced and written in Arabic. This kind of Arabic prokem used by students in Gontor is detailed in the table below. Examples of Using Prokem in Indonesian Context Sentences Examples of Using Prokem in Indonesian Context Sentences Meaning in Indonesian Language Arabic Prokem Page His style is funky, man! يتفنكف ن ك جيل 44 What else are they doing? ملاذا كمان ؟ 34 If you get hit by the caretaker, I’m also hit. إن كان أنت يضرب مع مدبر طيعا أنا يضرب كجكز 33 ARABIYAT, ISSN: 2356-153X, E-ISSN: 2442-9473 126 Arabic Prokem in Indonesian Vocabulary Arabic Prokem in Indonesian Vocabulary No Arabic Prokem Be read Meaning 1 صاع Sha’ Ostentatious 2 باص Bashi Fusty 3 مديك Mudik Laugh Arabic Prokem in Indonesian Vocabulary No Arabic Prokem Be read Meaning 1 صاع Sha’ Ostentatious 2 باص Bashi Fusty 3 مديك Mudik Laugh No Arabic Prokem Be read Meaning 1 صاع Sha’ Ostentatious 2 باص Bashi Fusty 3 مديك Mudik Laugh In the table above, the prokem “sha’” is written in Arabic but it actually comes from Indonesian, “sok” which means ostentatious. The “sok” is arabizationed through shad, alif and ‘ain which have almost the same sound. In Arabic vocabulary, there is no word “sa”. Next, “bashi” is prokem written and pronounced in Indonesian. This prokem means fusty. In Arabic, the same sound with this word is ba, alif and shad. There are four letters in Indonesian but three letters Arabic. This happens as the effect of language contrastive. Arabiyât Arabiyât Arabiyât 5 سيه Sih Affirmation 6 هيه Heh Wonder Based on the table above, at least six Arabic prokem words are commonly used by students in Gontor. Actually, the interjection prokem is also popular for Indonesian people, but this prokem is generally found in the context of sentence (siyâq al-kalâm) in Arabic and it is also written in Arabic. However, the meaning of the Arabic and Indonesian prokem in this form is slightly different. For example, “ih” in Arabic prokem means wonder but disappointed, disgusted, and scared. Then, “ah” in Arabic means disappointed but in Indonesian, its meaning is based on the context, it can be regret, disappointed, disagree or wonder. The following table presents examples of this prokem in a sentence. Table 7 Examples of Arabic Prokem in the Form of Interjection Meaning in Indonesian Language Arabic Prokem Page What’s wrong with you! كيف سيه أنت! 32 Don’t eat too much إيه ال ت كن تنظيف دائما 62 Table 7 Table 7 Examples of Arabic Prokem in the Form of Interjection Meaning in Indonesian Language Arabic Prokem Page What’s wrong with you! كيف سيه أنت! 32 Don’t eat too much إيه ال ت كن تنظيف دائما 62 Examples of Arabic Prokem in the Form of Interjection 4. Arabic Prokem from Arabi and Indonesian Free Morpheme Prokem, formed through Arabic and Indonesian free morpheme, is created as the effect of the mix of students’ native language to Arabic. Consequently, this prokem sounds interesting, funny, and unfamiliar. This prokem is like Indonesian-Arabic or Arabic-Indonesian, as seen in the table below. 3. Arabic Prokem in the Form of Interjection 3. Arabic Prokem in the Form of Interjection Arabic prokem in the form of interjection used by students in Gontor is a kind of feeling expression influenced by native language. Dialect is another influence of this prokem. The Arabic prokem in the form of interjection is shown in this following table. Table 6 Arabic Prokem in the Form of Interjection No Arabic Prokem Be read Meaning 1 آه Ah Disappointed 2 إيه Ih Wonder 3 بالحق Bi al-haq Swear 4 إيه Eh Surprise Table 6 Arabic Prokem in the Form of Interjection ARABIYAT, ISSN: 2356-153X, E-ISSN: 2442-9473 127 Arabiyât 5 سيه Sih Affirmation 6 هيه Heh Wonder Examples of Arabic Prokem with Indonesian Morpheme in Sentences Examples of Arabic Prokem with Indonesian Morpheme in Sentences Meaning in Indonesian Language Arabic Prokem Page Bagaimana sih kamu! كيف سيه أنت؟ 32 Eh jangan menghabiskan terus ! إيه ال نكن تنظيف دائما 62 Kamu sajalah نأ ت فقط اله 85 Kamu ngga apa-apa? Kok mukul saya sih أنت ال ماذا ماذا قو تضربنى سيه 119 Examples of Arabic Prokem with Indonesian Morpheme in Sentences Arabic Prokem from Arabic and Indonesian Free Morpheme No Arabic Prokem Be Read Meaning 1 خذت Khudztin Ambilin 2 كمطرا Kematharan Kehujanan 3 فقظ اله Fakat lah Sajalah 4 ال ماذا ماذا Laa madza madza Tidak apa-apa The table above shows that the Arabic prokem is from Arabic and Indonesian free morpheme. The prokem “Khudztin” comes from Arabic “akhadza” which means take, then it gets “tin” which is Indonesian morpheme. Originally, that Arabic language is a command word from “akhdaz”, referring to “khudz”. “Khudztin” is Arabic prokem from “khudz” and “tin”. It relates to the Indonesian morpheme that “in” indicates command words, such as ambilin. ARABIYAT, ISSN: 2356-153X, E-ISSN: 2442-9473 128 Arabiyât Another prokem influenced by Indonesian language is “kematharan”, which is from Arabic “mathar” which means rain. Then, there is “faqath lah” from “fakath” which means only. The morpheme “lah” is from Indonesian words, “sajalah”. Next, there is “la madza-madza” from Arabic “la” which means no and “mada” which menas what. In Indonesian language, it means never mind. Besides, Arabic prokem with Indonesian morpheme can be seen in sentences, as following table. Table 9 Examples of Arabic Prokem with Indonesian Morpheme in Sentences 5. Arabic Prokem in Reduplication Arabic prokem in the form of reduplication relates to repeated verbal and noun words. This prokem connects to Indonesian language, doubling the words to indicate plural meaning. The Arabic prokem in this case can be seen in the table below. ARABIYAT, ISSN: 2356-153X, E-ISSN: 2442-9473 Examples of Arabic Prokem in the Form of Reduplication Examples of Arabic Prokem in the Form of Reduplication Meaning in Indonesian Language Arabic Prokem Page Diam-diam berbahaya سكوت– سكوت ضرر 66 Orangnya besar-besar lagi إنسانه ك ب ير– كبير كمن 67 Sudahlah jangan banyak- banyak خالص اله ال تكن كثيرا كثير 113 Makan-makan yuk أكل– أكل يوك 112 Examples of Arabic Prokem in the Form of Reduplication Meaning in Indonesian Language Arabic Prokem Page Diam-diam berbahaya سكوت– سكوت ضرر 66 Orangnya besar-besar lagi إنسانه ك ب ير– كبير كمن 67 Sudahlah jangan banyak- banyak خالص اله ال تكن كثيرا كثير 113 Makan-makan yuk أكل– أكل يوك 112 Table 10 Table 10 Arabic Prokem in the Form of Reduplication Arabic Prokem Be read Meaning in Indonesian Language طريق- طريق Thariq-thariq Jalan-jalan أكل- أكل Akl- akl Makan-makan صعب- صعب Sa’b-sa’b Susah-susah سكوت- سكوت Syukut-syukut Diam-diam مرة- مرة Marrah-marrah Kali-kali كبير– كبير Kabir-kabir Besar-besar Table 10 Arabic Prokem in the Form of Reduplication Arabic Prokem Be read Meaning in Indonesian Language طريق- طريق Thariq-thariq Jalan-jalan أكل- أكل Akl- akl Makan-makan صعب- صعب Sa’b-sa’b Susah-susah سكوت- سكوت Syukut-syukut Diam-diam مرة- مرة Marrah-marrah Kali-kali كبير– كبير Kabir-kabir Besar-besar Arabic Prokem in the Form of Reduplication ARABIYAT, ISSN: 2356-153X, E-ISSN: 2442-9473 129 Arabiyât The table above shows how Arabic words are combined with Indonesian structure in repeating similar words. This reduplication form creates Arabic prokem. In Indonesian, repeated similar words are shown plural, such as rumah-rumah (houses). This structure is applied by students in Gontor in Arabic prokem, like thariq-thariq. In Arabic, thariq means walk (jalan). The prokem happens due to reduplication by repeating thariq to be thariq-thariq. Actually, walking around in Arabic is “masyyan”. This form is also applied to other prokem listed in the previous table. Examples of Arabic prokem in forms of sentence used by Gontor’s students can be seen in the following table. Examples of Using Arabic Prokem in the Form of Phoneme Change Meaning in Indonesian Language Arabic Prokem Page Biasaya kamu ke mana? آدة أنت إلينا 32 Saya juga tidak tahu أناكجك ال أعرف 44 Maaf Pak! أفا بك 44 Ente Bagus sekali! أنت ثقة جدا 75 Meaning in Indonesian Language Arabic Prokem Page Biasaya kamu ke mana? آدة أنت إلينا 32 Saya juga tidak tahu أناكجك ال أعرف 44 Maaf Pak! أفا بك 44 Ente Bagus sekali! أنت ثقة جدا 75 6. Arabic Prokem in the Form of Phoneme Change The phoneme change is also found in Arabic prokem used by students in Gontor. In this case, there is a phoneme change in the Arabic original word so that the Arabic word is no longer the same as the real one. This kind of Arabic phoneme can be in the form phoneme reduction, phoneme addition and phoneme change, as written in the table below. Table 12 Arabic Prokem in the Form of Phoneme Change No Arabic Prokem Be Read Meaning 1 كجك Kajaka Likewise 2 هجا Haja This 3 أفا Affan Apology 4 آدة Aadatan Usually 5 جلك Jalika That 6 قالص Qalash Already 7 سقة Siqqah True Arabic Prokem in the Form of Phoneme Change ARABIYAT, ISSN: 2356-153X, E-ISSN: 2442-9473 130 Arabiyât Through the table above, it can be explained several Arabic prokem through the change of phoneme. “Kajaka” is Arabic prokem from Arabic word “kadzaalik‟ which means “likewise”. This prokem has the phoneme change and reduction. The reduction is “lam” and the change is “dza” to “jim”. In Gontor, people use to have “kajaka” as prokem in daily life. Then, “haja” as Arabic proke actually is not found in Arab fushhâ. Its original word is “hadza” which means “this”. There is a change of phoneme in that prokem, from “dza” to “jim”. Next, “affan” as Arabic prokem comes from “„afwan”, which means my apology. The prokem was caused by the change of sound from the sound of "wau" to the sound of "fa", so that it became Arabic with double sound (tasydid). Another Arabic prokem is “adah” to “adah” that has phoneme change from “ain” to “hamzah”, but the meaning is still the same. Then, “dzalika” is from “jalika”. The phoneme change in this word is “dza” to “jim”. Next, there is qalas” from Arabic word, “khalas”. The phoneme change is “kha” to “ghin”. Finally, the Arabic prokem “siqqah” which comes from Arabic word, “tsiqqah”. The phoneme change is the sound of “tsa” to be “sa”. In the examples of sentences, the prokem can be seen as follow. Table 13 Examples of Using Arabic Prokem in the Form of Phoneme Change Arabiyât Arabiyât 5 مصدره Mashdaruh The source 6 جاكته Jaketuh The jacket Basically, Arabic prokem in the form of dhamîr muttashil through mufrad mudzakkar is not in accordance with the Arabic grammatical structure. For example, “hadzauh” is prokem that is incorrect in Arabic structure for some reasons. First, that prokem is from ism isyârah “Hadza”. Second, the plural form for isim israyah for masculin ((mudzakkar) is “haulai”, the plural is not used “wau”, just like in “hadauuh”. Third, ism isyârah cannot be linked to dhamîr muttashil. Therefore, “hadzauh” as prokem actually comes from Indonesian word “ini” and “nya” as the third pronoun, so this prokem means “ininya”. This condition also can be found in other prokem, such as “dzalikuh”, “insanuh”, “iddamuh”, “mashdaruh”, and “jakituh”. These prokem are adapted by Indonesian structure which is incorrect in Arabic structure. The Arabic prokem in the form of sentences can be seen in the thable below. Examples of Using Arabic Prokem with Dhamîr Muttashil Examples of Using Arabic Prokem with Dhamîr Muttashi Meaning in Bahasa Indonesia Arabic Prokem Page Ininya di mana? هذاؤه أين؟ 40 Dia bagaimana Orangnya? إنسانه كيف هو؟ 67 Dia sumbernya هو مصدره 70 Itunya rusak ذالكه فاسد 73 Lauknya sangat tidak enak غير جدا إدّمه 81 Kamu sok nyama-nyamain jaket. أنت يهان سواء كمان جاكته؟ 34 Examples of Using Arabic Prokem with Dhamîr Muttashi Meaning in Bahasa Indonesia Arabic Prokem Page Ininya di mana? هذاؤه أين؟ 40 Dia bagaimana Orangnya? إنسانه كيف هو؟ 67 Dia sumbernya هو مصدره 70 Itunya rusak ذالكه فاسد 73 Lauknya sangat tidak enak غير جدا إدّمه 81 Kamu sok nyama-nyamain jaket. أنت يهان سواء كمان جاكته؟ 34 Based on the previous analysis, it can be indicated that all of the tables show Arabic prokem and the way to use it in sentences. Overall, all the prokem is influenced by Indonesian language, in the form of dialect, grammatical structure, and vocabulary. This finding aligns with what Nugrawiyati (2014) said: prokem language's function has transitioned from secret language to slang. In Kamus Besar Bahasa Indonesia (as cited in Heru & Rukiyah, 2019), it states “slang is a variety of informal, non-standard, seasonal languages used by teens or members of particular social groups for internal communication with the goal that others will not understand”. 7. Arabic Prokem in the Form of Dhamîr Muttashil Arabic prokem in the form of dhamîr muttashil is created throuh one Arabic morpheme plus one Arabic pronoun (Dhamîr Muttashil). The dhamîr muttashil used is the third pronoun in the form of mufrad mufdzakkar (singular for masculine). Singular for feminine usually comes to this pronoun. This form of prokem can be seen in the following table. Arabic Prokem in the Form of Dlamir Muttasil Arabic Prokem in the Form of Dlamir Muttasil No Arabic Prokem Be Read Meaning 1 هذاؤه Hadzauh This 2 ذلكه Dzalikuh That 3 إنسانه Insanuh The person 4 إدّمه Iddamuh The dish ARABIYAT, ISSN: 2356-153X, E-ISSN: 2442-9473 131 Arabiyât 5 مصدره Mashdaruh The source 6 جاكته Jaketuh The jacket Arabiyât Conclusion Based on the analysis, it can be concluded that the causes of Arabic prokem used by students in Gontor are native grammatical tendency of the students. The students’ native language is Bahasa Indonesia. This process creates Arabic prokem which is only understood by students in Gontor. In fact, there is a small probability for outsider to understand this prokem without some explanation by the students or some information in research, including this conducted study. The study results show several forms and types of Arabic prokem used by students in Gontor. First, Arabic prokem in context of Indonesian language (Indonesian Arabic). Second, Arabic prokem in Indonesian vocabulary. Third, Arabic prokem in the form of interjection. Fourth, Arabic prokem from Arabic and Indonesian free morpheme. Fifth, Arabic prokem in reduplication. Sixth, Arabic prokem in the form of phoneme change. Seventh, Arabic prokem in the form of dhamîr muttashil. Arabiyât Slang is a sort of non- standard language that is still evolving in Indonesia with the intention of creating intimacy with group members, therefore it will likely always exist (Sulaeman, 2019, p. 53). By mixing words to create new meanings, slang creates a large number of new terms for the language (Fromkin et al in Rumagit & Rambing, 2020, p. 3) as found in the data above. ARABIYAT, ISSN: 2356-153X, E-ISSN: 2442-9473 132 REFERENCES Abdul Chair., & Leonie, A. (2014). Sosiolinguistik Perkenalan Awal. Jakarta: Rineka Cipta. Aryanti, Y., Indarti, M. D., & Priyanto, A. (2019). Analisis penggunaan bahasa prokem melalui media sosial whats application (wa) pada siswa sma. 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https://openalex.org/W2101095566	https://www.scielo.br/j/jbchs/a/R5nZkWjJWQkrDPyn4SCLPtx/?lang=en&format=pdf	English	null	Infra-red heating as an alternative technique for fast sample preparation	Journal of the Brazilian Chemical Society	2,000	cc-by	3,556	Infra-red Heating as an Alternative Technique for Fast Sample Preparation Sandro T. Gouveiaa, Orlando Fatibello-Filhob and Joaquim A. Nóbregab* aDepartamento de Química Analítica e Físico-Química, Universidade Federal do Ceará, Fortaleza - CE, Brazil bDepartamento de Química, Universidade Federal de São Carlos, CP 676, 13560-970, São Carlos - SP, Brazil aDepartamento de Química Analítica e Físico-Química, Universidade Federal do Ceará, Fortaleza - CE, Brazil bDepartamento de Química, Universidade Federal de São Carlos, CP 676, 13560-970, São Carlos - SP, Brazil Neste trabalho investigou-se um procedimento de preparo de amostras, com rápido aquecimento da suspensão da amostra em meio ácido usando radiação infravermelho emitida por lâmpadas de tungstênio. As vantagens do procedimento proposto são a simplicidade e o baixo custo. A solução diluída do ácido é aquecida em tempo inferior a 60 s e o tempo total do preparo da amostra é menor que 5 min. O procedimento proposto pode ser facilmente implementado para a análise de elementos presentes em baixas concentrações. Avaliações preliminares foram feitas determinando-se os teores de Cu, Fe, Mn e Zn em material de referência certificado de fígado bovino e folha de maçã. Todos os resultados foram avaliados com nível de confiança de 95%. Os teores determinados de Cu e Mn foram comparados em amostra de fígado bovino sem tratamento prévio ou após liofilização. Para essa amostra, os resultados foram validados empregando-se procedimento de decomposição assistida por microondas em frasco fechado. A procedure for sample preparation was investigated in this work using infra-red radiation generated by tungsten lamps for fast heating of an acidic sample suspension contained in a glass flask. The main advantages of the developed procedure are its simplicity and low-cost. Additionally the dilute acid solution is heated in less than 60 s and the total sample preparation is performed in less than 5 min. The proposed procedure is suitable for trace analysis and it can be easily implemented. The preliminary investigations focus on the determination of Cu, Fe, Mn, and Zn in bovine liver and apple leaves as standard reference materials. All results are in agreement at a 95% confidence level. Copper and Mn were also determined in a sample of bovine liver without any sample pretreatment or after a freeze-drying step for comparison. For this sample, the results were validated using a conventional microwave-assisted closed vessel procedure. Keywords: Infra-red heating; sample preparation; infra-red radiation; sample decomposition e-mail: djan@zaz.com.br J. Braz. Chem. Soc., Vol. 11, No. 3, 261-265, 2000. J. Braz. Chem. Soc., Vol. 11, No. 3, 261-265, 2000. c 2000 Soc. Bras. Química 0103 - 5053 $6.00+0.00 J. Braz. Chem. Soc., Vol. 11, No. 3, 261-265, 2000. Printed in Brazil Article Reagents, samples and apparatus Bovine liver samples with masses varying from 200 to 1400 mg were employed for evaluating the effect of sample masses on the decomposition using the tungsten lamp system. The decomposition was evaluated using 1.0 ml of HNO3 conc. plus H2O2 conc. solution with vol- umes varying from 0 to 1.0 ml in 0.5 ml aliquots. After- wards, the volume of the H2O2 conc. solution was fixed in 1.0 ml and the volume of the HNO3 conc. solution varied from 0.5 to 1.5 ml in 0.5 ml aliquots. For validation of the obtained results for Cu and Mn, these samples were also decomposed using a microwave-assisted procedure. In this case the sample mass of 350 mg was transferred to a Teflon perfluoroalkoxy (PFA) vessel and to which was added 3.0 ml HNO3 conc. and 0.5 ml H2O2. The heating program was implemented in 4 successive steps with a total cycle of 11 min (Table 1). All reagents were analytical reagent grade and all solu- tions were prepared using Milli-Q water (Millipore, 18 mΩ cm-1, USA). Hydrogen peroxide and HNO3 concentrated (Mallinckrodt, Germany) were used as received. Diluted solu- tions of Cu, Fe, Mn, and Zn were prepared from monoelement stock solutions containing 1,000 mg l-1 of each metals (Merck, Germany). The decomposition was carried out in a 20 ml boro- silicate glass flask with a PTFE cap. Several standard reference materials (SRM) from the National Institute of Standards and Technology (NIST) were employed during this investigation. Most experi- ments dealt with one biological reference material (bo- vine liver, SRM 1577b, NIST, USA) and one botanical reference material (apple leaves, SRM 1515, NIST, USA). Four bovine liver samples were employed as re- ceived to evaluate the performance of the procedure. For validating the results obtained with the proposed procedure, these samples were also acid decomposed employing a microwave-assisted closed vessel (Mile- stone, ETHOS 1600, Germany). Table 1. Microwave decomposition program. Step Time (min) Power (W) 1 1.0 250 2 1.0 0 3 4.0 250 4 5.0 400 In all digested solutions the determination of Cu and Mn was carried out by graphite furnace atomic ab- sorption spectrophotometry (GFAAS, SpectrAA 800, Varian, Australia) with a Zeeman background corrector. Iron and Zn were determined in the SRM’s by flame atomic absorption spectrophotometry (FAAS, SpectrAA 640, Varian, Australia). Introduction the performance of microwave-assisted procedures by mea- suring and controlling the temperature and the pressure and therefore improving safety. The analytical sciences have rapidly evolved with re- spect to the development of modern techniques for measur- ing inorganic and organic analytes in liquid medium. The sample preparation step still needs improvement and this is most critical for samples that contain a chemically resistant matrix, either organic or inorganic, with analytes present in low concentrations. The employment of other radiations, such as ultravio- let2 and ultrasound3,4, has also been proposed for sample preparation purposes. Ultraviolet radiation is well-estab- lished for decomposition of organic compounds in enviromental samples5. Infra-red radiation could be more extensively used based on its ability to promote fast heating of solutions. Infra-red radiation is usually employed for drying samples in cleaner conditions and for the preparation of sub-boiling acids6. Another interesting application of infra-red is the heating of liquid or solid samples for determination of volatile analytes7. This procedure was successfully applied for de- termination of iodine in milk samples8. The use of microwave radiation as a heating source is well-established in analytical chemistry and a plethora of procedures were established based on microwave-assisted digestion1. There is no doubt that these procedures are useful and that they paved a new method for sample prepa- ration. Modern instrumentation allows improvement of Some less conventional applications of infra-red radiation in analytical chemistry were proposed by Campos et al.9 and 262 Gouveia et al. J. Braz. Chem. Soc Figure 1. Infra-red decomposition arrangement. The third lamp (not shown) is positioned behind the sample flask. by Magalhães et al.10. Both groups used an arrangement with three tungsten lamps for direct analysis of solid samples. The former authors employed this system to burn organic samples and the volatile elements were transported by a gas flow carrier towards a flame AAS9. In the other investigation this device was used to volatilize Hg from sediment samples10. In this work three 150 W tungsten lamps configured in a triangular arrangement were employed to heat a glass flask containing the sample. The proposed procedure was evaluated for determination of Cu, Fe, Mn and Zn in a botanical and in a biological sample. Figure 1. Infra-red decomposition arrangement. The third lamp (not shown) is positioned behind the sample flask. Procedure Reagents, samples and apparatus Reagents, samples and apparatus Results and Discussion Table 4. Effect of the decomposition mixture on solubilization of Cu and Mn by infrared procedures. Table 4. Effect of the decomposition mixture on solubilization of Cu and Mn by infrared procedures. HNO3 (ml) H2O2 (ml) Copper (µg g-1) Manganese (µg g-1) 1.0 0 16.4 ± 0.3 2.76 ± 0.03 1.0 0.5 18.7 ± 0.1 2.98 ± 0.01 1.0 1.0 16.1 ± 0.5 2.75 ± 0.01 0.5 1.0 17.9 ± 1.3 2.78 ± 0.17 1.0 1.0 16.1 ± 0.5 2.75 ± 0.01 1.5 1.0 18.3 ± 0.7 2.57 ± 0.05 All initial studies were performed with raw bovine liver samples without any sample pre-treatment. The first experiment involved the investigation of the effect of sample mass on the solubilization of Cu and Mn using 1.0 ml HNO3 conc. plus 0.5 ml H2O2 conc. The obtained results are shown in Table 3. For sample masses less than 1000 mg there is a good agreement between the results for Cu and Mn despite a gradual trend to decreas- ing values. For the largest mass evaluated (1400 mg) the results for Cu and Mn were 19.4 and 9.8%, respectively, lower than the medias values of the previous analyses. In addition to the heating capacity and the efficiency of the heating transfer of the W lamps system, the decomposi- tion of high mass samples was also affected because of the use of a constant volume of the decomposition mix- ture. The experiment was carried out by applying 10 V to the W lamps, which is 22% of the total power supported by the 3 lamps connected in series. According to data presented in the literature with an analogous system10, the temperature in the focal point was 450oC when 10 V was applied. These same authors mentioned that the ap- plication of 40 V generated a temperature of 1700oC in the focal point. The temperatures were not measured, but it could be presummed that an unknown degree of over- heating could happen in the focal point. It was experi- mentally observed that concentrated HNO3 solutions The next step was the establishment of the effect caused by the heating time on the results for Mn in bo- vine liver. For a preliminary evaluation of the accuracy, this experiment was carried out using a mass of 30 mg of a SRM containing 10.5 ± 1.7 mg g-1. Reagents, samples and apparatus Masses varying from 30 to 200 mg of each SRM were transferred to the glass vials and the following procedure was applied: an aliquot of 1.0 ml of HNO3 conc. was added to each vial containing up to 100 mg of the SRM and the heating was promoted by applying a voltage of 10 V dur- ing 150 s. For masses greater than 100 mg, 2.0 ml of HNO3 conc. was added and the heating was promoted by apply- ing 10 V during 240 s. The flask was carefully opened and volumes of 0.5 or 1.0 ml of H2O2 conc. solution were added. The same voltage was then applied during 90 s. The flask containing the decomposition mixture and the sample was A schematic diagram of the infra-red heating system is shown in Figure 1. Three 15 V-150 W infra-red lamps (Osram, Halogen-Bellaphort, Germany) were electrically connected in series. The lamps were set in a triangular arrangement to form a common focal point for all the lamps. They were connected to a Variac transformer (SP Sociedade Técnica Paulista LTDA, Brazil). Vol. 11 No. 3, 2000 Vol. 11 No. 3, 2000 Infra-red Heating as an Alternative Technique for Fast Sample Preparation 263 Table 3. Effect of sample mass on the solubilization of Cu and Mn by infrared procedures. Mass (mg) Copper (µg g-1) Manganese (µg g-1) 200 18.1 ± 0.2 2.90 ± 0.1 500 18.0 ± 1.1 2.89 ± 0.1 1000 17.9 ± 1.3 2.78 ± 0.2 1400 14.5 ± 0.1 2.58 ± 0.0 Table 3. Effect of sample mass on the solubilization of Cu and Mn by infrared procedures. positioned in the focal point of the triangular lamp ar- rangement. The resulting suspensions were quantitatively transferred to 25 ml volumetric flasks, without any previ- ous centrifugation step and the volumes were made up with water. Copper and Mn were determined by GFAAS using the standard additions method. The heating program developed is shown in Table 2. Due to their greater concentrations, Fe and Zn were determined by FAAS using air-C2H2 and the conditions recommended by the manufacturer11. boiled in 40 s in a closed vessel when a voltage of 10 V was applied. To investigate the effect of the decomposition mixture on the solubilization of the Cu and Mn, the sample mass was kept constant in 1000 mg and the proportion of HNO3 conc. and H2O2 conc. Reagents, samples and apparatus was varied according to data shown in Table 4. Applying the Q test at a 95% confidence level for the results obtained for both elements it can be con- cluded that all results are acceptable and there is no pro- nounced effect of the decomposition mixture in the range evaluated. Unless when mentioned, all further experiments were carried out using 1.0 ml HNO3 conc. and 0.5 ml H2O2 conc. According to the literature the use of H2O2 increased the concentration of oxalate generated during decomposi- tion due to the partial decomposition of more complex or- ganic compounds12. However, this is not so critical taking into account that all measurements were performed by GFAAS. Table 2. Graphite furnace heating program. Step Temp (oC) Time (s) Gas flow rate Read (l min-1) 1 85 5.0 3.0 No 2 95 40.0 3.0 No 3 120 10.0 3.0 No 4 800 1000 5.0 3.0 No 5 800 1000* 1.0 3.0 No 6 800 1000* 2.0 0.0 No 7 2300 2800* 1.1 0.0 Yes 8 2300 2800* 2.0 0.0 Yes 9 2500 2800* 2.0 3.0 No * Temperature for Manganese Table 2. Graphite furnace heating program. Results and Discussion It can be concluded that the solubilization of Mn occurs rapidly in this SRM and the heating time is not a critical parameter. All re- sults shown in Table 5 with heating times varying from 2 to 15 min led to accurate results for Mn in bovine liver. All further experiments were carried out using a 2 min heating time. For larger masses, the heating time was pro- portionally incremented. The content of Cu in this SRM was also measured for validation purposes and for a preliminary check of the applicability of the procedure for other elements, the con- tents of Fe and Zn were also determined (Table 6). Even for Fe, a difficultty soluble element13, there is a good agree- ment between determined and certified values. Gouveia et al. J. Braz. Chem. Soc 264 Table 5. Effect of heating time in the determination of Mn in bovine liver. (Certified value is 10.5 ± 1.7 µg g-1, SRM 1577b, NIST). Time (min) Mn (µg g-1) 2 9.8 ± 0.2 4 9.9 ± 0.1 8 9.1 ± 0.3 10 10.1 ± 0.2 15 9.2 ± 0.2 Both decomposition procedures, i.e. infrared and micro- waves, were implemented. Considering the results obtained and the typical water content of raw bovine liver (70%)14, it was found a complete agreement between the results for both elements after freeze-drying or without any previous treatment was found. The results are shown in Table 8. The ratio obtained between raw and freeze-dried values for each element are close to the ratio between the raw and the freeze-dried sample masses (3.3). It can be concluded that the particle size was not so critical for bovine liver and the solubilization of the analytes was effective even for roughly cut raw samples. For further evaluation of the applicability of the infra- red procedure, Cu, Fe, Mn, and Zn were also determined in a botanical standard reference material (apple leaves). Bo- tanical samples can be more difficultt to decompose than biological samples due to the formation of insoluble sili- cate compounds in the vegetal tissue13. For all elements apart from Zn, slightly lower values were determined when compared to certified values; the worst was that obtained for Fe which is not surprising for a botanical matrix owing to the formation of mixed oxides with Si that require HF for complete solubilization. Conclusions The infra-red heating procedure was successfully ap- plied for determination of Cu, Mn, and Zn in bovine liver and apple leaves. The procedure was also suitable for the determination of Fe in bovine liver, but the results for apple leaves were lower than expected. The behavior of different analytes present in different matrices needs to be fully evalu- ated to establish advantages and disadvantages of the infra- red procedure. At this point, its main characteristics are the low cost and the fast sample preparation. The partial de- composition can be an obstacle for some analytical tech- niques of measurement. We intend to further develop this procedure as an one flask procedure for preparation of low mass samples for trace analysis. Additionally, it can be men- tioned that instead of using the US$ 120.00 lamps, with the tungsten coil inserted in a gold concave structure, US$ 5.00 halogen lamps (Decostar 51, 50 W - 12 V, Osram, Germany) could be used but the heating speed is reduced 3-fold. Three bovine liver samples were also decomposed adopting a microwave-assisted procedure. The results com- paring this procedure and the infrared procedure for Cu and Mn are shown in Table 7. Despite an apparent positive systematic error for Cu values determined using the IR procedure, a paired t-test showed that all results for Cu and Mn are in agreement at a 95% confidence level. All measurements were performed without any sample pre-treatment. To evaluate the effect of water content and particle sizes on the infrared decomposition procedure, one of the previous bovine liver samples was freeze-dried and Cu and Mn were determined in the resulting powder. Table 6. Determination of Cu, Fe, Mn, and Zn in botanical and biological standard reference materials. Mean values and SDs (n = 5). Bovine Liver Apple Leaves Certified Determined Certified Determined Cu (mg g-1) 160 ± 8 155 ± 10 5.64 ± 0.24 5.17 ± 0.65 Mn (mg g-1) 10.5 ± 1.7 9.0 ± 0.4 54 ± 3 41 ± 2 Fe (mg g-1) 184 ± 5 172 ± 8 80* 42.4 ± 0.2 Zn (mg g-1) 127 ± 16 126 ± 19 12.5 ± 0.3 13.8 ± 0.7 Table 8. Effect of water content and particle sizes on the infrared decomposition procedure. Results and Discussion Despite this small deviation between measured and certified values, Cu, Mn, and Zn results are in agreement at a 95% confidence level. Infra-red Heating as an Alternative Technique for Fast Sample Preparation Vol. 11 No. 3, 2000 265 Acknowledgements 5. Zlotorzynski, A. Crit. Rev. Anal. Chem. 1995, 25, 43. 6. Howard, A. G.; Statham, P. J. Inorganic Trace Analysis Philosophy and Practice; John Wiley, Chichester, 1993. O.F.F. and J.A.N. are grateful to Conselho Nacional de Desenvolvimento Científico e Tecnológico for research fellowships. S.T.G. would like to express his gratitude to PICDT/CAPES for the fellowship. We are grateful to Fundação de Amparo à Pesquisa do Estado de São Paulo for research funds (1997/4323-4 and 98/10814-3). 7. Knaap, G.; Raptis, E.; Kaiser, G.; Tölg, G.; Scharmel, P.; Schreiber, B. Fresenius Z. Anal. Chem. 1981, 308, 97. 8. Gélinas, Y.; Krushevska, A.; Barnes, R. M. Anal. Chem. 1998, 70, 1021. FAPESP helped in meeting the publication costs of this article. Conclusions M W IR Lamps Raw Freeze-dried f Raw Freeze-dried f Cu (µg g-1) 48.3 ± 2.6 144.5 ± 13.8 3.0 52.6 ± 5.2 164.1 ± 4.6 3.1 Mn (µg g-1) 1.44 ± 0.01 4.53 ± 0.27 3.1 1.53 ± 0.09 5.10 ± 0.28 3.3 Table 7. Comparison of digestion methods for bovine liver samples. Sample Copper (µg g-1) Manganese (mg g-1) M W IR Lamps M W IR Lamps 1 52.8 ± 3.8 69.7 ± 0.9 2.49 ± 0.30 2.36 ± 0.22 2 137.2 ± 7.4 148.6 ± 13.0 1.30 ± 0.06 1.34 ± 0.10 3 48.3 ± 2.6 52.6 ± 5.2 1.44 ± 0.01 1.53 ± 0.09 *Not Certified Table 8. Effect of water content and particle sizes on the infrared decomposition procedure. M W IR Lamps Raw Freeze-dried f Raw Freeze-dried f Cu (µg g-1) 48.3 ± 2.6 144.5 ± 13.8 3.0 52.6 ± 5.2 164.1 ± 4.6 3.1 Mn (µg g-1) 1.44 ± 0.01 4.53 ± 0.27 3.1 1.53 ± 0.09 5.10 ± 0.28 3.3 Table 8. Effect of water content and particle sizes on the infrared decomposition procedure. Infra-red Heating as an Alternative Technique for Fast Sample Preparation References 9. Campos, R. C.; Curtius, A. J.; Berndt, H. J. Anal. At. Spectrom. 1990, 5, 669. 10. Magalhães, C. E. C.; Krug, F. J.; Fostier, A. H.; Berndt, H. J. Anal. At. Spectrom. 1997, 12, 1231. 1. Kingston, H. M.; Haswell, S. J. Microwave-enhanced Chem- istry. 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https://openalex.org/W1987039033	https://hal.archives-ouvertes.fr/hal-00951283/document	English	null	Classification of clouds sampled at the puy de Dôme (France) based on 10 yr of monitoring of their physicochemical properties	Atmospheric chemistry and physics	2,014	cc-by	19,967	Classification of clouds sampled at the puy de Dôme (France) from 10-year monitoring: Mean features of their physico-chemical properties. Laurent Deguillaume, Tiffany Charbouillot, Muriel Mourguy Joly, Mickaël Vaïtilingom, Marius Parazols, Angela Marinoni, Pierre Amato, A.M. Delort, V. Vinatier, Andrea Flossmann, et al. To cite this version: Laurent Deguillaume, Tiffany Charbouillot, Muriel Mourguy Joly, Mickaël Vaïtilingom, Marius Para- zols, et al.. Classification of clouds sampled at the puy de Dôme (France) from 10-year monitoring: Mean features of their physico-chemical properties.. Atmospheric Chemistry and Physics, 2014, 14 (3), pp.1485-1506. 10.5194/acp-14-1485-2014. hal-00951283 Distributed under a Creative Commons Attribution 4.0 International License Classiﬁcation of clouds sampled at the puy de Dôme (France) based on 10 yr of monitoring of their physicochemical properties L. Deguillaume1,2, T. Charbouillot1,2,3,4, M. Joly1,2,3,4, M. Vaïtilingom1,2,3,4, M. Parazols1,2,3,4, A. Marinoni1,2,, P. Amato3,4, A.-M. Delort3,4, V. Vinatier3,4, A. Flossmann1,2, N. Chaumerliac1,2, J. M. Pichon1,2, S. Houdier5,6, P. Laj1,2,, K. Sellegri1,2, A. Colomb1,2, M. Brigante3,4, and G. Mailhot3,4 1 1Clermont Université, Université Blaise Pascal, OPGC, Laboratoire de Météorologie Physique, BP 10448, 63000 Clermont-Ferrand, France 2CNRS, UMR6016, LaMP/OPGC, BP80026, 63177 Aubière, France 3 3Clermont Université, Université Blaise Pascal, Institut de Chimie de Clermont-Ferrand, BP 10448, 63000 Clermont-Ferrand, France , 4CNRS, UMR6296, ICCF, BP 80026, 63177 Aubière, France 5Université Joseph Fourier-Grenoble 1, Laboratoire de Glaciologie et Géophysique de l’Environnement, 38041 Grenoble France 6CNRS, UMR5183, 38041 Grenoble, France now at: the Institute for Atmospheric Science and Climate (ISAC), CNR, Bologna, Italy * now at: the Institute for Atmospheric Science and Climate (ISAC), CNR, Bologna, Italy now at: Laboratoire de Glaciologie et Géophysique de l’Environnement (LGGE), CNRS/University of Grenoble, Grenoble, France p ( ) g y now at: Laboratoire de Glaciologie et Géophysique de l’Environnement (LGGE), CNRS/University of Grenoble, Grenoble, France Correspondence to: L. Deguillaume (l.deguillaume@opgc.univ-bpclermont.fr) Correspondence to: L. Deguillaume (l.deguillaume@opgc.univ-bpclermont.fr) Received: 23 July 2013 – Published in Atmos. Chem. Phys. Discuss.: 30 August 2013 Revised: 6 December 2013 – Accepted: 6 January 2014 – Published: 10 February 2014 Received: 23 July 2013 – Published in Atmos. Chem. Phys. Discuss.: 30 August 2013 Revised: 6 December 2013 – Accepted: 6 January 2014 – Published: 10 February 2014 tion. Total organic carbon (TOC) is signiﬁcantly higher in polluted air masses than in the other categories, which sug- gests additional anthropogenic sources. Concentrations of carboxylic acids and carbonyls represent around 10 % of the organic matter in all categories of air masses and are stud- ied for their relative importance. Iron concentrations are sig- niﬁcantly higher for polluted air masses and iron is mainly present in its oxidation state (+II) in all categories of air masses. Finally, H2O2 concentrations are much more varied in marine and highly marine clouds than in polluted clouds, which are characterized by the lowest average concentration of H2O2. This data set provides concentration ranges of main inorganic and organic compounds for modeling purposes on multiphase cloud chemistry. Abstract. Long-term monitoring of the chemical composi- tion of clouds (73 cloud events representing 199 individual samples) sampled at the puy de Dôme (pdD) station (France) was performed between 2001 and 2011. Physicochemical pa- rameters, as well as the concentrations of the major organic and inorganic constituents, were measured and analyzed by multicomponent statistical analysis. Classiﬁcation of clouds sampled at the puy de Dôme (France) based on 10 yr of monitoring of their physicochemical properties L. Deguillaume1,2, T. Charbouillot1,2,3,4, M. Joly1,2,3,4, M. Vaïtilingom1,2,3,4, M. Parazols1,2,3,4, A. Marinoni1,2,, P. Amato3,4, A.-M. Delort3,4, V. Vinatier3,4, A. Flossmann1,2, N. Chaumerliac1,2, J. M. Pichon1,2, S. Houdier5,6, P. Laj1,2,**, K. Sellegri1,2, A. Colomb1,2, M. Brigante3,4, and G. Mailhot3,4 1 Along with the corre- sponding back-trajectory plots, this allowed for distinguish- ing four different categories of air masses reaching the sum- mit of the pdD: polluted, continental, marine and highly ma- rine. The statistical analysis led to the determination of cri- teria (concentrations of inorganic compounds, pH) that dif- ferentiate each category of air masses. Highly marine clouds exhibited high concentrations of Na+ and Cl−; the marine category presented lower concentration of ions but more el- evated pH. Finally, the two remaining clusters were classi- ﬁed as “continental” and “polluted”; these clusters had the second-highest and highest levels of NH+ 4 , NO− 3 , and SO4− 2 , respectively. This unique data set of cloud chemical com- position is then discussed as a function of this classiﬁca- HAL Id: hal-00951283 https://hal.science/hal-00951283v1 Submitted on 5 Sep 2020 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License 1 Introduction The atmospheric system is a complex, dynamical, and mul- tiphase medium where chemical species can be transformed (Monks et al., 2009). Different phases (solid, gas, liquid and ice) are in permanent interaction; here, the multiphase cloud system holds a particular interest. It contains both organic and inorganic species that result from the mass transfer of soluble gases in the liquid phase and also from the dissolution of the soluble fraction of aerosol particles (Fuzzi et al., 2002; Sellegri et al., 2003). Modeling studies (e.g., Barth, 2006; Leriche et al., 2007; Lim et al., 2005; Tilgner and Herrmann, 2010; Tost et al., 2007), in situ measurements (e.g., Laj et al., 1997b; Möller et al., 1996; Moore et al., 2004) and lab- oratory investigations (e.g., Altieri et al., 2006; Vione et al., 2006) highlighted the fact that chemical reactions in clouds play an important role in atmospheric chemistry and strongly affect the homogeneous and heterogeneous gas-phase chem- istry (Lelieveld and Crutzen, 1991). Depending on the phys- ical and chemical characteristics of cloud droplets, aqueous- phase reactions potentially represent either a sink or a source for atmospheric trace compounds. In turn, the chemical trans- formations in the liquid phase control the fate of gaseous species and/or their reaction products upon cloud dissipation. Transport, distribution and removal of chemical species from the atmosphere are clearly inﬂuenced by physical and chem- ical processes in clouds. It is therefore crucial to study the chemical composition and reactivity of chemical species in clouds in order to un- derstand and quantify their mechanisms of transformation in the atmosphere. Several in situ investigations of cloud chem- istry have already been reported, and most of them are based on speciﬁc single campaigns (Bower et al., 2000; Choular- ton et al., 1997; Collett Jr et al., 1990; Decesari et al., 2005; Elbert et al., 2000; Ghauri et al., 2001; Gioda et al., 2009; Hegg et al., 2002; Hutchings et al., 2009; Kim et al., 2006a; Löﬂund et al., 2002; Moore et al., 2004; Sedlak et al., 1997; Watanabe et al., 2001; Weathers et al., 1988; Wieprecht et al., 2005; Wilkinson et al., 1997; Wobrock et al., 2001). All of these studies showed a high variability in the cloud chemical composition, which is driven by both source proximity and local microphysics. 1 Introduction Integration studies considering different cloud experiments can be difﬁcult, especially because cloud sampling methodologies are not standardized and not easily comparable (Laj et al., 2009). Some long-term ﬁeld measure- ments using automated cloud water collection systems were conducted in the past; they mainly examined the inorganic chemical composition of clouds (Acker et al., 1998; Baum- gardner et al., 2003; Guo et al., 2012; Kim et al., 2006b). The role of clouds in the atmospheric cycle of inorganic substances has been discussed in many publications in the past (see recent reviews from Fowler et al., 2009; Monks et al., 2009) focusing on the oxidation/transformation of sul- fur and nitrogen compounds (Barth, 2006; Cape et al., 1997; Seinfeld and Pandis, 2006; Wells et al., 1997). The impor- tance of the atmospheric liquid phase has been investigated for organic substances (Fuzzi et al., 2002; Herckes et al., 2013), in particular for the formation/transformation of car- boxylic acids (Chebbi and Carlier, 1996; Laj et al., 1997a; Monod and Carlier, 1999) or, more recently, for the forma- tion of secondary organic aerosol (SOA) after cloud evapo- ration (Blando and Turpin, 2000; Hallquist et al., 2009; Lee et al., 2012; Lim et al., 2010; Loefﬂer et al., 2006). Actu- ally, although the formation of SOA has been largely stud- ied in the gas phase, the contribution of clouds is still un- certain (Ervens et al., 2011). Complex chemical reactions can occur in aerosol water, forming accretion products such as oligomers. Recent laboratory studies demonstrated that small oxygenated compounds undergo these processes such as methylglyoxal (Altieri et al., 2008; Tan et al., 2010), hy- droxyacetaldehyde (Perri et al., 2009) or glyoxal (Carlton et al., 2007; De Haan et al., 2009; Ervens and Volkamer, 2010); these alternative chemical pathways are assumed to represent an efﬁcient process to transform volatile organic compounds into SOA (El Haddad et al., 2009). For modeling purposes, statistical information on the vari- ability of cloud chemical composition and its relationship with environmental factors, such as source regions, is ex- tremely useful. Long-term studies are required to derive sta- tistically sound information. Due to the transient nature of clouds, monitoring cloud chemical composition is not triv- ial and is subject to difﬁculties such as sampling frequen- cies, samples’ conservation, and quality homogeneity on a long-term basis. L. Deguillaume et al.: Classiﬁcation of clouds sampled at the puy de Dôme (France) 1486 produced oxidants such as hydroxyl and nitrate radicals (re- spectively, HO q and NO q 3) as well as oxidants such as ozone (O3) and hydrogen peroxide (H2O2). Reactivity is also in- ﬂuenced by the partitioning between droplets and the other phases (particle, gas, ice) and is catalyzed by photochem- ical processes (Barth, 2006; Hoigné, 1994; Minero et al., 2007; Ruggaber et al., 1997; Vione et al., 2006). Recently, a number of studies also suggested that biological processes are potentially involved in the transformation of soluble or- ganic material in cloud droplets, even at low temperatures (Deguillaume et al., 2008; Husárová et al., 2011; Vaïtilingom et al., 2010, 2011, 2013). An additional complexity derives from the presence of ice, which interferes with liquid/gas phase partitioning and modiﬁes the photochemical equilib- rium through feedbacks between ice, radiation and reactivity pathways (Long et al., 2010; Marécal et al., 2010; Voisin et al., 2000). Published by Copernicus Publications on behalf of the European Geosciences Union. Published by Copernicus Publications on behalf of the European Geosciences Union. L. Deguillaume et al.: Classiﬁcation of clouds sampled at the puy de Dôme (France) 2.1 Cloud sampling Sampling was performed at the puy de Dôme (pdD) sta- tion (45◦46′20′′ N, 2◦57′57′′ E), which is part of the net- works EMEP (the European Monitoring and Evaluation Pro- gramme), GAW (Global Atmosphere Watch), and ACTRIS (Aerosols, Clouds, and Trace gases Research Infrastructure). The observatory chalet is on top of a mountain rising notably above the surrounding area with an altitude of 1465 m a.s.l.; the pdD mountain is part of the Massif Central mountains, a topography that is north–south oriented. Clouds are fre- quently observed at the top of the site, either during ad- vection of frontal systems or because of the orographic ris- ing of moist air. The urban area of Clermont-Ferrand and its surrounding suburbs (≈280 000 inhabitants) is situated 16 km east of the station at 396 m a.s.l.; its inﬂuence on the pdD measurements is fairly limited (especially during night- time) and air masses producing clouds at the pdD summit from eastern origin are extremely rare, as documented by pri- mary anthropogenic tracers concentrations (NO, black car- bon, etc.) and statistical studies on aerosol size distributions (Asmi et al., 2012; Henne et al., 2010). Most of the clouds are sampled during winter and spring and their corresponding air masses are representative of various geographical origins. A short summary of previous studies related to this site is given in the Supplement. After collection, cloud water samples were kept at 4 ◦C in the dark until the chemical and total organic carbon (TOC) analyses were conducted. TOC was obtained with a Shi- madzu TOC 5050A analyzer. Potassium hydrogen phthalate and sodium carbonate aqueous solutions were used to cali- brate the instrument for the total carbon content (TC) and the inorganic carbon content (IC). TOC quantiﬁcation was then obtained by the difference between the measured TC and IC. The analytical method for ion chromatography analysis (ma- terial: Dionex DX320, column AS11 for anions, eluent KOH; Dionex ICS1500, column CS16 for cations, eluent hydrox- ymethanesulfonate) has been previously reported by Jaffrezo et al. (1998). Ion chromatography analysis of tenfold diluted samples was made in triplicate or more, and allowed for the measurement of the concentrations of the major organic and inorganic ions (acetic, formic, succinic, malonic and oxalic acids, Cl−, NO− 3 , SO2− 4 , Na+, K+, NH+ 4 , Mg2+ and Ca2+). Total ion content (TIC) corresponds to the calculated sum of the concentrations of these species. 1 Introduction Here, we present chemical data of the con- densed phase of a series of cloud events (73 cloud events representing 199 individual samples) sampled at the puy de Dôme (pdD) mountain, a high-altitude site (1465 m) that can generally be classiﬁed as “rural” (Henne et al., 2010; Joly and Peuch, 2012; Putaud et al., 2004). The observation of clouds and, more generally, of atmospheric variables at high- altitude, remote sites brings additional valuable information Chemical reactions in clouds are driven by efﬁcient ox- idation processes due to the presence of photo-chemically Atmos. Chem. Phys., 14, 1485–1506, 2014 www.atmos-chem-phys.net/14/1485/2014/ L. Deguillaume et al.: Classiﬁcation of clouds sampled at the puy de Dôme (France) 1487 paction (supercooled conditions) or were collected directly as a liquid. The water was transferred at room temperature, either directly or after a short melting period into glass vials. Possible changes in the cut-off diameter due to the formation of ice on the impaction plate are expected to be negligible given the limited volume of the collected sample. The accu- mulation of ice leads to more efﬁcient sampling by collecting smaller drops. on the extent of the anthropogenic inﬂuence. Usually, these sites are inﬂuenced by long-range transport rather than by local sources, and hence provide information on the physic- ochemical variability of air masses on a regional scale. More precisely, due to the high altitude of the station, the air mass composition is representative of the regional atmo- spheric background during the day and more representative of larger synoptic-scale air masses during nighttime (Asmi et al., 2011). The data set presented (pH, conductivity, redox potential, total organic content, concentrations of the most abundant inorganic and organic chemical species, concentra- tions of oxidants: iron and hydrogen peroxide) could be used as a reference for modeling work that simulates the chemical composition of tropospheric clouds in different scenarios. 2.2 Physicochemical parameters and chemical analysis Physicochemical parameters were measured immediately af- ter sampling; pH, conductivity and redox potential were mea- sured using a Consort® multi-parameter C830. Meteorologi- cal parameters were also monitored at the pdD station: wind speed and direction, temperature, pressure, and relative hu- midity. Measurements of liquid water content (LWC) and ef- fective radius of droplets were obtained from a Gerber PVM- 100 probe, which was regularly controlled and calibrated us- ing a spinning optical disk. 2.1 Cloud sampling Measurements of H2O2 and iron concentrations by UV– visible spectroscopy were done in triplicate or more; the derivatization of these reactive species in the samples al- lowed for maintaining their concentrations until the analysis was performed in the laboratory; this was directly conducted after the end of the collection of cloud water. H2O2 concen- tration was measured by UV–visible spectroscopy at 551 nm, following the method developed by Bader et al. (1988). This method is based on the oxidation of N,N-diethyl- p-phenylenediamine (DPD) by H2O2 in the presence of horseradish peroxidase, leading to the radical cation of DPD with an absorbance (at 551 nm) of 21 000 ± 500 M−1 cm−1 at equimolar rate. Fe(II) concentration was measured by UV– visible spectroscopy at 562 nm, using the method developed by Stookey (1970) based on the rapid complexation of iron with ferrozine. After reduction of Fe(II) by the addition of Cloud sampling was performed between 2001 and 2011, with an average sampling time of 2 h (corresponding to an average sampling volume of 50 mL). However, between 2006 and 2009, the observatory at the pdD summit has been ren- ovated, which greatly limited the cloud sampling operations (only 16 cloud events were sampled during that period). Non- precipitating cloud droplets were sampled using a single- stage cloud collector similar to the one described by Kruisz (1993). Cloud droplets larger than 7 µm (cut-off diameter) (Brantner et al., 1994) were collected by impaction onto a rectangular aluminum plate. Droplets either froze upon im- L. Deguillaume et al.: Classiﬁcation of clouds sampled at the puy de Dôme (France) hydroxylamine chlorhydrate, the total iron content Fe(tot) was detected. Fe(III) concentration was then calculated by deducing the concentration of Fe(II) from Fe(tot). were declared different when the probability for the groups to have identical data distribution was lower than 5 % (p value < 0.05). This test was chosen due to the lack of nor- mality of the data according to the Shapiro–Wilk normality test. were declared different when the probability for the groups to have identical data distribution was lower than 5 % (p value < 0.05). This test was chosen due to the lack of nor- mality of the data according to the Shapiro–Wilk normality test. Measurements of carbonyl compounds were done by us- ing a method that was speciﬁcally developed for the quantiﬁ- cation of single aldehydes in environmental aqueous phases (Houdier et al., 2000). The method, which has been re- cently improved for the analysis of polyfunctional carbonyls (Houdier et al., 2011), is based on the derivatization of car- bonyl compounds with an original oxyamino reagent, i.e., dansylacetamidooxyamine (DNSAOA). Oximes that form in solution are subsequently separated and detected by using high performance liquid chromatography (HPLC) with ﬂu- orescence detection. The method exhibits limits of detection below 1 nM for the compounds targeted here, i.e., formalde- hyde (HCHO), acetaldehyde (CH3CHO) and polyfunctional carbonyls glyoxal (CHOCHO), in addition to methylglyoxal (CH3COCHO) and hydroxyacetaldehyde. HOCH2CHO and the analyses of the carbonyl content of cloud samples were tripled for a quantitative approach. Samples were frozen un- til quantiﬁcation of carbonyls, which was done once a year. Previous works highlighted the full recovery of carbonyls af- ter samples were frozen, since they are analyzed right after their thawing (Domine et al., 2010; Houdier et al., 2011). In cloud droplets, carbonyl compounds (in particular formalde- hyde) form adducts with dissolved SO2. For instance, sig- niﬁcant amounts of hydroxymethanesulfonate (HMSA), the adduct of HCHO, have been measured in cloud or fog wa- ter where it constitutes a reservoir for both S(IV) species and HCHO (Ang et al., 1987; Munger et al., 1984). HMSA and more generally S(IV)-carbonyl adducts form rapidly and are stable towards dissociation in acidic media such as cloud droplets (Dasgupta et al., 1980; Munger et al., 1984). In this work, derivatization of carbonyls by DNSAOA was performed in an acidiﬁed (pH ∼2) solution of the cloud sample. 3 Results Seventy-three cloud events have been sampled at the top of the pdD between 2001 and 2011, representing 199 individ- ual samples. The cloud chemical composition as well as the physicochemical parameters measured at the pdD station can be found on a website (http://wwwobs.univ-bpclermont.fr/ SO/beam/data.php). Table S1 indicates the physicochemical analysis performed for each cloud event together with the origin of the air masses reaching the sampling site. For this study, we decided to classify our cloud samples ac- cording to a statistical analysis that considers physicochem- ical parameters of the cloud samples. This approach is more robust than considering only the back-trajectories of the air masses that reach the puy de Dôme station. Nevertheless, the back-trajectories are drawn for each cloud samples to con- ﬁrm the statistical classiﬁcation. Cloud events correspond- ing to 4 individual samples (No. 19 and 27, Table S1) with air masses originating from the Sahara according to the cor- responding back-trajectory plots were excluded due to their lack of representativeness of the data set. 2.4 Back-trajectory plots Seventy two-hour backward trajectories of the air masses col- lected at the puy de Dôme were calculated using the HYS- PLIT (Hybrid Single-Particle Lagrangian Integrated Trajec- tory) model with the GDAS1 meteorological data archive and default settings (Draxler and Rolph, 2012). L. Deguillaume et al.: Classiﬁcation of clouds sampled at the puy de Dôme (France) Dissociation of the carbonyl-S(IV) adducts in the course of the derivatization reaction is therefore unlikely, and carbonyl concentrations reported in this paper represent the free carbonyl concentrations ([carb]free) and therefore lower estimates of the total carbonyl concentrations, with [carb]tot = [carb]free+ [S(IV)-carb]. 3.1 Origin of air masses – climatology Individual samples were classiﬁed by principal component analysis (PCA). This technique was previously applied to inorganic ion concentrations in cloud water and to the air mass origin (Deininger and Saxena, 1997; Kim et al., 2006b). Here, this statistical approach is used to distinguish the pa- rameters that inﬂuence the cloud sample characteristics. www.atmos-chem-phys.net/14/1485/2014/ Atmos. Chem. Phys., 14, 1485–1506, 2014 1488 2.3 Statistical analyses (B) Variables factor map: loadings of the 6 experimental variables (pH, [Na+], [Cl−], [SO2− 4 ], [NO− 3 ], [NH+ 4 ]). 1489 -3 -2 -1 0 1 2 3 4 5 -4 -3 -2 -1 0 1 2 3 4 5 6 7 PC2 = 0.30 Group 1 = Highly Marine Group 4 = Polluted Group 3 = Continental Group 2 = Marine -0.2 -0,1 -0.0 0.1 0.2 0.3 0.4 0.5 0.6 -0.15 -0.10 -0.05 -0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55 pH Na+ (µM) Cl- (µM) SO4 2- (µM) NH4 + (µM) NO3 - (µM) PC1 = 0.50 PC1 = 0.50 PC2 = 0.30 B) Variables factor map A) Individuals factor map Fig. 2. Air masses reaching the puy de Dôme are divided into four distinct back-trajectory sectors: west (W), northwest/north (NW/N), northeast (NE) and south/southwest (S/SW). Percentages in black represent the occurrence of cloud events in each sector. Percentages in dark blue, light blue, green and red represent the occurrence of cloud events in each sector with highly marine, marine, continental and polluted inﬂuences, respectively. Fig. 2. Air masses reaching the puy de Dôme are divided into four distinct back-trajectory sectors: west (W), northwest/north (NW/N), northeast (NE) and south/southwest (S/SW). Percentages in black represent the occurrence of cloud events in each sector. Percentages in dark blue, light blue, green and red represent the occurrence of cloud events in each sector with highly marine, marine, continental and polluted inﬂuences, respectively. Fig. 1. (A) Individuals factor map: scores of the 138 cloud sam- ples (i.e., 56 cloud events) on the plan PC1-PC2 obtained from the six selected variables. The different colors indicate the hierarchi- cal classiﬁcation using the Ward method. This led to the constitu- tion of 4four clusters corresponding to polluted, continental, marine and highly marine categories. (B) Variables factor map: loadings of the 6 experimental variables (pH, [Na+], [Cl−], [SO2− 4 ], [NO− 3 ], [NH+ 4 ]). mining the relevant criterion to distinguish marine and conti- nental inﬂuence. For each cluster, the mean, maximal and minimal values of the variables used for the statistical analysis were calculated; these values were used as criteria to classify cloud events that have not been considered in the PCA analysis. These crite- ria are summarized in Table 1. 2.3 Statistical analyses Principal component analysis (PCA) was performed using Simca-P + software (Umetrics). Air mass categories were determined with a “hierarchical clustering analysis” (HCA) from PCA results: cloud events or groups of cloud events were lumped together in pairs based on their similarities. In our analysis, only one missing value from the six variables (pH and Na+, Cl−, NO− 3 , NH+ 4 and SO2− 4 concentrations) used for the PCA was permitted to limit artifacts. At ﬁrst, 13 variables measured at the pdD station were considered in the analysis: major ion concentration (Na+, Cl−, NO− 3 , NH+ 4 and SO2− 4 ), total ion content, cloud wa- ter loading, liquid water content, pH, conductivity, redox po- tential, iron concentration and total organic carbon. Only the six variables that discriminated between the different groups most were kept to perform a second PCA: pH and Na+, Cl−, NO− 3 , NH+ 4 and SO2− 4 concentrations. Among all cloud events, only 138 samples, corresponding to 56 events, pre- sented sufﬁcient data for the analysis (one missing value at maximum). The two ﬁrst PCs (principle components) of To highlight signiﬁcant differences between data groups, the Kruskal–Wallis nonparametric test was carried out with Past software (Hammer et al., 2001). Two air mass categories www.atmos-chem-phys.net/14/1485/2014/ Atmos. Chem. Phys., 14, 1485–1506, 2014 L. Deguillaume et al.: Classiﬁcation of clouds sampled at the puy de Dôme (France) L. Deguillaume et al.: Classiﬁcation of clouds sampled at t -3 -2 -1 0 1 2 3 4 5 -4 -3 -2 -1 0 1 2 3 4 5 6 7 PC2 = 0.30 Group 1 = Highly Marine Group 4 = Polluted Group 3 = Continental Group 2 = Marine -0.2 -0,1 -0.0 0.1 0.2 0.3 0.4 0.5 0.6 -0.15 -0.10 -0.05 -0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55 pH Na+ (µM) Cl- (µM) SO4 2- (µM) NH4 + (µM) NO3 - (µM) PC1 = 0.50 PC1 = 0.50 PC2 = 0.30 B) Variables factor map A) Individuals factor map Fig. 1. (A) Individuals factor map: scores of the 138 cloud sam- ples (i.e., 56 cloud events) on the plan PC1-PC2 obtained from the six selected variables. The different colors indicate the hierarchi- cal classiﬁcation using the Ward method. This led to the constitu- tion of 4four clusters corresponding to polluted, continental, marine and highly marine categories. 2.3 Statistical analyses Among the last 57 samples not considered in this statistical analysis, 12 were classiﬁed as highly marine, 25 as marine, 7 as continental, and 13 as polluted. this second analysis represented more than 80 % of the to- tal variability of the samples (PC1: 50.41 %; PC2: 30.08 %) (Fig. 1a), i.e., 80 % of the disparity between groups is kept despite the fact that only two axes are used to represent six variables. In order to highlight the inﬂuence of the air mass ori- gin on cloud droplet chemical composition, events were divided into four different sectors of origin according to the corresponding back-trajectory plots: west (W), north- west/north (NW/N), northeast (NE) and south/southwest (S/SW) (Fig. 2). Cloud events from the West and the north- west/north sectors were the most frequent ones, representing 72 % of the air masses sampled at the pdD station, the major- ity of which was categorized as “marine” or “highly marine”. Air masses originating from west were mainly under Atlantic Ocean inﬂuence (80 % of the events from the west) while the air masses originating from the northwestern/northern sec- tor could also be subject to continental effects from United Kingdom (UK) (24 % of the events from NW/N were clas- siﬁed as continental). The northeastern sector was character- ized by air masses with high anthropogenic inﬂuence due to eastern Europe emissions (55 % of the events were polluted). Air masses from the southwest/south sector were dominated The hierarchical clustering analysis led to the constitution of 4four clusters. The ﬁrst one, classiﬁed as “highly marine”, is characterized by high concentrations of Na+ and Cl−(with means of 311 and 232 µM, respectively) while for the second group, classiﬁed as “marine”, a low concentration of these ions is found. For both clusters (“marine” and “highly ma- rine”), the pH range is elevated and similar. Finally, the two remaining clusters were classiﬁed as “continental” and “pol- luted”; these clusters had the second-highest and highest lev- els of NH+ 4 , NO− 3 , and SO4− 2 , respectively. Despite the fact that the four categories are well separated, they partly overlap (Fig. 1a). Hence, air mass back-trajectories (not taken into account in the analysis presented on Fig. 1a) have also been considered for establishing this categorization. This also con- ﬁrms that the tricky point for the selection is clearly deter- Atmos. Chem. Phys., 14, 1485–1506, 2014 L. Deguillaume et al.: Classiﬁcation of clouds sampled at the puy de Dôme (France) 1490 Table 1. Mean, minimum and maximum values for each discriminating parameter (pH, concentrations of Cl−, Na+, NO− 3 , SO2− 4 , NH+ 4 ) for the different categories of cloud events (polluted, continental, marine and highly marine) determined by the PCA analysis. Concentrations (µeq L−1) pH Cl− Na+ NO− 3 SO2− 4 NH+ 4 Polluted Mean 4.0 142 115 451 159 357 Min 3.8 29 86 368 147 332 Max 4.2 203 172 516 171 376 Continental Mean 4.9 31 31 105 94 142 Min 3.9 5 6 32 26 73 Max 7.0 87 99 298 218 340 Marine Mean 5.7 30 32 24 28 43 Min 4.7 1 1 1 3 4 Max 7.6 231 316 93 78 96 Highly marine Mean 6.2 232 311 72 73 96 Min 4.7 110 138 24 33 48 Max 6.9 394 679 232 144 190 such as Jungfraujoch (3450 m a.s.l., Switzerland, Bal- tensperger et al., 1998) or Sonnblick (3106 m a.s.l., Austria, Hitzenberger et al., 2000). Polluted air masses sampled at the pdD station are the most acidic (mean pH = 4.3 ± 1.1) and present signiﬁcantly higher conductivity than the other types of air mass. TIC values are also higher for polluted and highly marine conditions due to the higher amount of nitrate, sulfate and ammonium for polluted air masses and sodium and chloride for highly marine clouds. These observations are conﬁrmed by other studies (Cini et al., 2002; Collett et al., 2002; Kim et al., 2006a; Möller et al., 1996). For example, Collett et al. (2002) sampled cloud water on the west coast of the USA and at Mount Whiteface and Mount Mitchell. The average pH on the west coast was around 6.0, because of the marine inﬂuence of the Paciﬁc Ocean, while the pH at Mount Whiteface and Mount Mitchell, two sites located close to ur- ban areas, was very acidic (up to 2.4) (Collett et al., 2002). Regarding the work of Aleksic et al. (2009) at the Whiteface mountain during 12 yr from 1994 to 2006, no signiﬁcant pH variation (close to 4) was reported over this period. Our cloud water samples present naturally well-correlated conductivity and TIC (R = 0.92); polluted air masses are more acidic and have the highest amount of ionic species; consequently, they present a high conductivity and TIC (Kim et al., 2006a). L. Deguillaume et al.: Classiﬁcation of clouds sampled at the puy de Dôme (France) by a pronounced marine character (64 %) and a continental inﬂuence from Spain (36 %). During the past 10 yr, in total 13 % of the air masses sampled at the pdD station were under highly marine inﬂuence, 52 % were under marine inﬂuence, 26 % were under continental inﬂuence, and 9 % were under anthropogenic inﬂuence. p g In the next part of this paper, we will focus on the variabil- ity of the chemical composition of cloud water as a function of air mass categories determined by our statistical analy- sis. Table S2 presents the physicochemical parameters (pH, redox potential, conductivity, liquid water content) and con- centrations of measured chemical compounds (average, min- imum, maximum) as a function of air mass origins together with data from recent studies describing chemical composi- tion of clouds. Since the chemical makeup of clouds is suf- ﬁciently different from that of fog, primarily due to the en- vironments in which they form (Collett et al., 2002; Straub et al., 2012; Weathers et al., 1988), data for fog have been excluded here. Although these studies featured a variety of cloud collection devices, different analytical techniques, and often a limited number of samples, some general compar- isons can be made with our data set. www.atmos-chem-phys.net/14/1485/2014/ Atmos. Chem. Phys., 14, 1485–1506, 2014 L. Deguillaume et al.: Classiﬁcation of clouds sampled at the puy de Dôme (France) www.atmos-chem-phys.net/14/1485/2014/ L. Deguillaume et al.: Classiﬁcation of clouds sampled at the puy de Dôme (France) 1491 g p p y ( ) Fig. 3. Distribution of pH, total ion content (TIC), conductivity (C), redox potential (Eh), total organic carbon (TOC) and liquid water content (LWC) of the cloud water sampled at the pdD station for each air mass category. The number of samples analyzed is indicated above each box plot. The bottom and top lines correspond to 25th and 75th percentiles, respectively. The full line and the square symbol represent the median and mean values, respectively. The ends of whiskers are 10th and 90th percentiles. Statistical differences (Kruskal–Wallis test; p value < 0.05) between groups are indicated by symbols above box plots. Fig. 3. Distribution of pH, total ion content (TIC), conductivity (C), redox potential (Eh), total organic carbon (TOC) and liquid water content (LWC) of the cloud water sampled at the pdD station for each air mass category. The number of samples analyzed is indicated above each box plot. The bottom and top lines correspond to 25th and 75th percentiles, respectively. The full line and the square symbol represent the median and mean values, respectively. The ends of whiskers are 10th and 90th percentiles. Statistical differences (Kruskal–Wallis test; p value < 0.05) between groups are indicated by symbols above box plots. as Mg2+, Na+ and Ca2+ for example. In these cases, a subse- quent continental or anthropogenic component of metals can be due to their dissolution from anthropogenic particles (such as ashes emitted from power plants, from combustion engine exhaust or from industrial operations). The concentrations of dissolved iron at the pdD station conﬁrm this general state- ment (see Sect. 4.5). Some authors have even proposed the use of the iron concentration as a parameter for determining the redox potential in hydrometeors, which would be feasi- ble thanks to the limited range of pH and Eh in atmospheric aqueous samples (Sinner et al., 1994; Stumm and Morgan, 1996). possible to compare the oxidative capacities of cloud wa- ter from one event to the other. Figure 3 shows that the average redox potential, Eh, of cloud water in polluted air masses (122.3 mV) was signiﬁcantly higher than in marine and highly marine air masses (22.0 and 4.3 mV, respectively) (p values: polluted/marine = 4 × 10−3; polluted/highly ma- rine = 7.1 × 10−5). L. Deguillaume et al.: Classiﬁcation of clouds sampled at the puy de Dôme (France) It is also interesting to note that in pol- luted air masses, Eh is always positive, demonstrating that polluted clouds are oxidizing media in the atmosphere, while continental and marine cloud can be either an oxidizing or reducing medium. This observation could be the result of the higher abundance of transition metal ions (TMI) commonly measured in cloud water originating from polluted air mass. TMI, i.e., Fe, Mn, Cu, etc., present a higher standard redox potential than the species typically found in marine air such TOC is signiﬁcantly higher in polluted air masses (with an average value of 12.4 mgC L−1) than in continental, ma- rine and highly marine ones (5.5, 3.3, and 4.8 mgC L−1, 3.2 Physicochemical parameters The redox potential of a water solution characterizes the average capacity of dissolved material to gain or lose elec- trons and reﬂects the reactivity. Many factors can limit the interpretation of the redox values measured in sampled cloud water such as temperature and pH. The redox poten- tial also depends on the concentrations of reduced and ox- idized forms of a large number of redox couples in solu- tion, inﬂuencing their reactivity and equilibria in cloud wa- ter. However, if a direct interpretation of the redox poten- tial data is not possible in natural environments, it is still Table 2 summarizes the minimum, maximum, average and median values of TIC, pH, conductivity, redox potential, TOC and LWC for the whole data set; Table S2 shows the minimum, maximum and average values of the physico- chemical parameters for each category of air mass, and Fig. 3 represents the distribution of these parameters as a function of air mass categories. g TIC values are relatively low and characteristic of Eu- ropean high-altitude sites, far from local pollution sources, www.atmos-chem-phys.net/14/1485/2014/ Atmos. Chem. Phys., 14, 1485–1506, 2014 3.3 Inorganic ions The relative distribution of inorganic ions was calculated for each single event; the average values of the different air mass categories are presented in Fig. 4. Only cloud events where all the inorganic ions have been quantiﬁed are considered (6, 46, 66, and 16 cloud samples for polluted, continental, ma- rine and highly marine categories, respectively). For a direct comparison of the four air mass categories, a radar chart plot presenting the relative contributions of each ion is also drawn in this ﬁgure. Table S2 shows the minimum, maximum, and average concentration values of inorganic ions calculated for all cloud events. The potential sources of nitrate, sulfate and ammonium in cloud water are multiple and include particles (ammonium sulfate and ammonium nitrate particles) and gases (from SO2, HNO3, NH3, N2O5) (Leaitch et al., 1988; Sellegri et al., 2003). The concentrations of ammonium, nitrate and sulfate are signiﬁcantly correlated within each category of air mass (R varies between 0.64 to 0.94, n = 6 to 70). This result can be attributed to the important contribution from nucleation scavenging of particles which prevails for nitrate, sulfate and ammonium to the overall cloud scavenging (Leriche et al., 2007). In our study, nitrate, sulfate and ammonium ions are abundant, as previously observed during studies at pdD (Sel- legri et al., 2003) and during other ﬁeld campaigns (Aleksic et al., 2009; Löﬂund et al., 2002); these represent an average contribution to the TIC of 65 % (marine), 76 % (continental) and 85 % (polluted). The highly marine clouds’ contribution to the TIC is much lower (25 %), due to the high concentra- tions of sodium and chloride; however, the absolute average concentrations of nitrate (59 µM), sulfate (79 µM) and ammo- nium (88 µM) ions are still rather elevated in this category of air mass. Observations made at the pdD station for marine and highly marine air masses are in agreement with those of Gioda et al. (2009) and Reyes-Rodriguez et al. (2009) who sampled air masses from Atlantic Ocean in Puerto Rico and showed similar pH and concentrations of inorganic ions. Blas et al. (2008) sampled cloud water at the “Black Triangle”, As already mentioned, the liquid water content (LWC) measured at the pdD station exhibits rather limited variation, with an average value of 0.28 ± 0.12 g m−3 (Fig. 3). L. Deguillaume et al.: Classiﬁcation of clouds sampled at the puy de Dôme (France) 1492 Table 2. Minimum, maximum, mean, and median values of physicochemical parameters (pH, conductivity (C), total ion content (TIC), total organic content (TOC), redox potential (Eh), liquid water content (LWC)) for the whole data set. Table 2. Minimum, maximum, mean, and median values of physicochemical parameters (pH, conductivity (C), total ion content (TIC), total organic content (TOC), redox potential (Eh), liquid water content (LWC)) for the whole data set. pH C TIC TOC Eh LWC (µS cm−1) (µeq L−1) (mgC L−1) (mV) (g m−3) Number of samples 143 90 140 106 93 105 Av 5.5 48 415 5 33 0.28 Min 3.1 2 19 1 −164 0.05 Max 7.6 348 1930 25 230 0.92 Med 5.6 25 335 3 35 0.27 respectively). In the case of polluted events, a further anthro- pogenic source of organic carbon can be superimposed on the background level. A few studies reported measurements of this parameter for cloud water (Table S3) and presented values within the same range as our data. A previous study at the pdD station from 2001 to 2002 reported DOC (dissolved organic carbon) values ranging from 1.2 to 15.5 mgC L−1 (Marinoni et al., 2004). These values of DOC are quite close to the TOC values reported here for the same sampling site, suggesting a lower contribution of the water-insoluble or- ganic carbon. Reyes-Rodriguez et al. (2009) measured the TOC and the DOC in cloud water from marine origin: the TOC ranged from 0.15 to 0.66 mgC L−1 and the DOC from 0.13 to 0.65 mgC L−1 (Reyes-Rodríguez et al., 2009) con- ﬁrming that a signiﬁcant fraction of TOC was composed of water-soluble organics (DOC/TOC = 0.79). Field measure- ments of fogs also reported that DOC constitutes about 80 % of the total organic carbon in the aqueous phase (Herckes et al., 2002a; Raja et al., 2008; Straub et al., 2012). A supple- mentary organic contribution is also observed in highly ma- rine clouds, which presented signiﬁcantly higher TOC values than marine clouds (p value = 4 × 10−4). tions of our classiﬁcation. To facilitate the comparison with other studies, we will present their concentrations expressed in µeq L−1 for the ions. www.atmos-chem-phys.net/14/1485/2014/ Atmos. Chem. Phys., 14, 1485–1506, 2014 3.3 Inorganic ions Distribution of the ratio “nitrate to sulfate” concentrations (expressed in µeq L−1) of the cloud water sampled at the pdD sta- tion as a function of air mass categories. Numbers above each box plot represent the number of samples analyzed. At least three data are required to represent a box plot. The bottom and top of box plots are 25th and 75th percentiles, respectively. The full line and the square symbol represent the median and mean values, respectively. The ends of whiskers are 10th and 90th percentiles. Statistical dif- ferences (Kruskal–Wallis test; p value < 0.05) between groups are indicated by symbols above box plots. the pdD site from local sources and the efﬁcient oxidation of NOx compounds converted into nitric acid and then nitrate (Wang et al., 2011). The nitrate to sulfate equivalent ratio is shown on Fig. 5 for the four categories of clouds. This ra- tio is highest in polluted clouds (from 0.9 in highly marine air masses to 3.6 for polluted clouds) due to the high nitrate content in polluted air masses. Some studies showed that the NO− 3 / SO2− 4 ratio increases with the distance from the source of pollution, which makes this value an indicator of the re- moteness of a site (Wang et al., 2011; Weathers et al., 1988). At the pdD sampling site, since the origin of the air masses is variable with high nitrate fractions for polluted air masses, this conclusion cannot be applied. Several values of this ra- tio reported elsewhere for cloud water samples were below 1 (Baltensperger et al., 1998; Bridges et al., 2002) but the ob- servation of elevated NO− 3 relative to SO2− 4 in cloud water has been also reported in other studies (Brüggemann et al., 2005; Hayden et al., 2008; Hill et al., 2007; Leaitch et al., 1986; Lee et al., 2012). This could be related to high NOx emission and the predominance of nitrate over sulfate in the acidiﬁcation of aerosols. Fig. 4. Distribution of the major inorganic ions measured (Cl−, NO− 3 , SO2− 4 , Na+, K+, NH+ 4 , Mg2+ and Ca2+) for each air mass category (percentages were calculated using the concentrations of ions in µeq L−1). The sum of the concentrations of inorganic cations and anions in µeq L−1 is indicated, as well as its contribution to the TIC. 3.3 Inorganic ions Several studies discussed the relationship between LWC and solute inorganic concentrations (Aleksic and Dukett, 2010; Elbert et al., 2000) where nonlinear relationships were extracted and evaluated. These relationships reﬂect the fact that an increase in the LWC should lead to a dilution of the cloud solute con- tent. In our study, we see no relationships between the LWC and the inorganic solute concentrations. Therefore, the vari- ations observed in the solute concentrations are not due to variations in LWC (R = −0.08) and to dilution effect. For organic matter, we also observed no relationships between TOC and LWC (Fig. S1). The solute (both organic and in- organic) concentrations might be attributed to the various in- ﬂuences to which cloud droplets are subjected: microphysics, meteorology, gas-phase reactions, chemical reactions in the liquid phase, and sedimentation (Herckes et al., 2013). In the next sections, concentrations of inorganic, organic and oxidant species are presented and discussed as func- Atmos. Chem. Phys., 14, 1485–1506, 2014 www.atmos-chem-phys.net/14/1485/2014/ e puy de Dôme (France) 1493 Fig. 5. Distribution of the ratio “nitrate to sulfate” concentrations (expressed in µeq L−1) of the cloud water sampled at the pdD sta- tion as a function of air mass categories. Numbers above each box plot represent the number of samples analyzed. At least three data are required to represent a box plot. The bottom and top of box plots are 25th and 75th percentiles, respectively. The full line and the square symbol represent the median and mean values, respectively. The ends of whiskers are 10th and 90th percentiles. Statistical dif- ferences (Kruskal–Wallis test; p value < 0.05) between groups are indicated by symbols above box plots. L. Deguillaume et al.: Classiﬁcation of clouds sampled at the puy de Dôme (France) 1493 L. Deguillaume et al.: Classiﬁcation of clouds sampled at Fig. 4. Distribution of the major inorganic ions measured (Cl−, NO− 3 , SO2− 4 , Na+, K+, NH+ 4 , Mg2+ and Ca2+) for each air mass category (percentages were calculated using the concentrations of ions in µeq L−1). The sum of the concentrations of inorganic cations and anions in µeq L−1 is indicated, as well as its contribution to the TIC. The radar chart shows the relative contribution of each inor- ganic ion as a function of the air mass categories. Fig. 5. 3.3 Inorganic ions The radar chart shows the relative contribution of each inor- ganic ion as a function of the air mass categories. one of Europe’s most industrialized regions; pH and inor- ganic ions concentrations in anthropogenic cloud water sam- pled at pdD were in the same range. In France, measure- ments of cloud chemical composition in the Vosges moun- tains show high sulfate and nitrate concentrations attributed to an anthropogenic additional source (Herckes et al., 2002b; Lammel and Metzig, 1991). Such data are comparable to in- organic concentrations reported for polluted air masses at the puy de Dôme station. Ammonium in cloud water results from the precursor aerosol particles or from the dissolution of gaseous ammo- nia. Its contribution to the TIC is relatively high in all air mass categories. Therefore, the pH of cloud water is inﬂu- enced by ammonium related to emissions from agricultural activities. However, the sum of nitrate and sulfate concen- trations is much higher than the ammonium concentration for polluted cases, resulting in partial acid neutralization and acidic cloud water. The concentration of calcium is higher in The contribution of nitrate to the TIC (from 6 % in highly marine cloud water to 48 % in polluted clouds) measured at the pdD site is higher than in ﬁeld measurements at other lo- cations. A possible explanation could be the remoteness of 3.4 Carboxylic acids – carbonyl compounds Field investigations including recent observations aimed to characterize and quantify individual organic compounds in cloud water during speciﬁc ﬁeld campaigns with a focus on small chain organic compounds such as mono- and dicar- boxylic acids and some carbonyls and dicarbonyls (Herckes et al., 2013). Here, we present the concentrations of 5 car- boxylic acids and 5 carbonyls as a function of the air mass categories. Figure 6 shows the mean relative contribution of ﬁve car- boxylic acids (formic, acetic, oxalic, malonic and succinic acids) for each air mass category. Only the cloud events where all these compounds have been measured are con- sidered (2, 23, 31 and 14 samples for polluted, continental and marine and highly marine categories, respectively). Ta- ble S2 indicates the minimum, maximum, and average values of carboxylic acid concentrations calculated for all events. Acetic and formic acids represent the most abundant of the ﬁve carboxylic acids, as previously observed (Khwaja, 1995; Löﬂund et al., 2002). Carboxylic acids, in the atmospheric aqueous phase, have different sources, such as anthropogenic and biogenic direct emissions in the gas and particulate phases, or chemical transformations from organic precursors (Charbouillot et al., 2012; Chebbi and Carlier, 1996; Khare et al., 1999). Formic acid can result from the oxidation of methanol by radicals (mainly HO q during daytime condi- tions). Acetic acid can be produced in the aqueous phase by the oxidation of various alcohols, like ethanol, ethylene gly- col or 2-propanol (Tilgner and Herrmann, 2010). The trans- fer from the gas phase is also an important source of formic and acetic acids in the atmospheric aqueous phase, whose Henry’s law constants are equal to 5400 and 5300 M atm−1 respectively (Johnson et al., 1996). Moreover, the presence of acetic and formic acids can also be potentially the result of their production by microorganisms as they are common intermediates in different metabolic pathways (Husárová et al., 2011). Oxalic acid is the most abundant carboxylic acid after acetic and formic acids (Sorooshian et al., 2006). Oxalic acid is produced through several steps of oxidation. In con- trast to acetic and formic acids, oxalic acid in the aqueous phase does not originate from the gas phase, which can ex- plain its lower concentration. Moreover, it efﬁciently creates metallic complexes with iron, which represent a sink through photolysis (Deguillaume et al., 2005). L. Deguillaume et al.: Classiﬁcation of clouds sampled at the puy de Dôme (France) polluted and continental air masses than in marine air masses, as a result of the higher contribution of its terrestrial source rather than its marine source. For polluted air masses, it is possible that the Ca2+ and/or Mg2+ may have been present as nitrate salts (CaNO3, MgNO3) (R(Ca2+ / NO− 3 ) = 0.47; R(Mg2+/NO− 3 ) = 0.78) or sulfate salts (CaSO4, MgSO4) (R(Ca2+ / SO2− 4 ) = 0.67; R(Mg2+/SO2− 4 ) = 0.54) as sug- gested by Leaitch et al. (1986). For highly marine clouds, the water is enriched by salts formed from the evaporation of sea spray, such as CaCO3, leading to a high Ca2+ concentration. Ca2+ also contributes to the neutralization of the acidity, but to a smaller extent than ammonium. In marine air masses, the average ratio Cl−/ Na+ is equal to 1.41 and reaches 1.06 for clouds deﬁned as highly marine, a value that is below that of seawater (1.17; Holland, 1978). This loss of chlorine may be caused by the deposition of HCl gas to the sea surface be- fore its absorption by cloud droplets (Watanabe et al., 2001). Another explanation could be that the aerosol ages during its transport: in the aerosol phase, the reaction NaCl + HNO3 gives NaNO3 and HCl, which volatilizes, leading to a de- pletion of Cl−(Bourcier et al., 2012; Sørensen et al., 2005). For polluted air masses, this ratio is 2.54, resulting from the enrichment of chloride over polluted areas. In fact, chloride ions have a marine source from NaCl particles and also an anthropogenic source with the dissolution of gaseous HCl into the aqueous phase (McCulloch et al., 1999; Shapiro et al., 2007). 2 sented in Fig. 4, showing that the relative contribution of each inorganic ion is very similar for marine and continental air masses. By contrast, the polluted air masses exhibit much higher concentrations of nitrate in the cloud water, and the cloud water samples in highly marine air are characterized by relatively high levels of chloride and sodium. sented in Fig. 4, showing that the relative contribution of each inorganic ion is very similar for marine and continental air masses. By contrast, the polluted air masses exhibit much higher concentrations of nitrate in the cloud water, and the cloud water samples in highly marine air are characterized by relatively high levels of chloride and sodium. www.atmos-chem-phys.net/14/1485/2014/ Atmos. Chem. Phys., 14, 1485–1506, 2014 1494 L. Deguillaume et al.: Classiﬁcation of clouds sampled at the puy de Dôme (France) L. Deguillaume et al.: Classiﬁcation of clouds sampled at the puy de Dôme (France) 1495 HMG = 3.7 × 103, HHA = 4.1 × 104 M atm−1 at 25 ◦C) (Bet- terton and Hoffmann, 1988; Sander et al., 2006) was shown to yield formic and oxalic acids (Altieri et al., 2008; Carl- ton et al., 2007; Perri et al., 2009) but also, to a lesser ex- tent, succinic and malonic acids (Perri et al., 2009). Concen- tration levels for F, AC, GL and MGL have already been measured concomitantly in rain or cloud water (Igawa et al., 1989; Munger et al., 1995; Li et al., 2008; van Pinx- teren et al., 2005; Ervens et al., 2013), but only few stud- ies have accounted for HA concentrations (see for instance, Matsumoto et al., 2005; Matsunaga and Kawamura, 2000). Figure 7 presents the relative contributions of these ﬁve car- bonyls for the four air mass categories. The concentration (average, min, max) of the ﬁve carbonyl compounds for the whole data set are presented in Table 3. phase are supposed to come exclusively from the particle phase. At present, only a few studies have highlighted the production of succinic and malonic acids from oxidation pro- cesses in the cloud aqueous phase (Charbouillot et al., 2012; Perri et al., 2009). This lack of aqueous-phase reactivity as a potential source of succinic and malonic acids could ex- plain the lower concentrations of these acids in cloud water. They are also much less concentrated in the particulate phase than acetic, formic and oxalic acids (Legrand et al., 2007). Globally, concentrations of carboxylic acids that were mon- itored at the pdD station are similar to those measured at the Raxalpe mountains in Austria (Löﬂund et al., 2002) or at the Schmücke mountains in Germany (van Pinxteren et al., 2005) (see Table S2), which are both removed from local pollution. The concentration of these organic acids is higher for polluted clouds, demonstrating the supplemental sources for air masses resulting from their passage over polluted area. For highly marine clouds, the amount of these acids is on the same order as the one for continental clouds, showing a ma- rine input of organic matter as conﬁrmed by TOC measure- ments (O’Dowd et al., 2004). L. Deguillaume et al.: Classiﬁcation of clouds sampled at the puy de Dôme (France) , y p g By comparison with the TOC measurements, we can esti- mate the presence of other organic species in cloud water, like alcohols, carbonyl compounds, larger multifunctional structures that contain a signiﬁcant fraction of heteroatoms (S, N, O) (such as humic-like substances (HULIS), ful- vic material, organosulfur species) (Feng and Möller, 2004; Graber and Rudich, 2006; LeClair et al., 2012), and also bi- ologically derived material (Bauer et al., 2002; Delort et al., 2010; Ekström et al., 2010). The proportion of undetermined organic matter in cloud water is around 90 % for the four categories showing the large unidentiﬁed portion of organic matter in cloud water (Fig. 6, see text above each pie chart). Carbonyl compounds have also been monitored at the pdD station: formaldehyde (F) was measured since 2000, and ac- etaldehyde (AC), glyoxal (GL), methylglyoxal (MGL) and hydroxyacetaldehyde (HA) were also quantiﬁed for 24 cloud samples between 2008 and 2011. Carbonyls in cloud water essentially result from their dissolution from the gas phase into the aqueous phase according to their Henry’s law con- stants (Matsumoto et al., 2005). In the aqueous phase, the oxidation of carbonyls produces carboxylic acids (Legrand et al., 2007) and also potentially leads to the formation of oligomers (Ervens and Volkamer, 2010) which can con- tribute, upon cloud droplets’ evaporation, to SOA. Particu- larly interesting, with this scope in mind, is the key role of α-oxygenated carbonyls GL, MGL and HA measured in this study. Oxidation by the HO q radical of these highly water- soluble compounds (Henry ’s law constant HGL = 3.6 × 105; By comparison with the TOC measurements, we can esti- mate the presence of other organic species in cloud water, like alcohols, carbonyl compounds, larger multifunctional structures that contain a signiﬁcant fraction of heteroatoms (S, N, O) (such as humic-like substances (HULIS), ful- vic material, organosulfur species) (Feng and Möller, 2004; Graber and Rudich, 2006; LeClair et al., 2012), and also bi- ologically derived material (Bauer et al., 2002; Delort et al., 2010; Ekström et al., 2010). The proportion of undetermined organic matter in cloud water is around 90 % for the four categories showing the large unidentiﬁed portion of organic matter in cloud water (Fig. 6, see text above each pie chart). 3.4 Carboxylic acids – carbonyl compounds Finally, of the quan- tiﬁed acids, succinic and malonic acids are the least abun- dant in all air mass categories. Opposite to acetic and formic acids, succinic and malonic acids in the atmospheric aqueous As the marine ratio for SO2− 4 / Na+ is known (0.06 on a molar basis; Holland, 1978), the contribution of SO2− 4 from anthropogenic sources, i.e., non sea-salt sulfate (nss- SO2− 4 ), can be inferred. The contribution of nss-SO2− 4 ranges from 86 to 100 % of the SO2− 4 dissolved in polluted clouds, whereas for marine clouds, this value is more varied and ranges from 5 to 99 %, with an average of 89 %. For highly marine clouds, the mean contribution of nss-SO2− 4 is much lower and reaches 47 %. This demonstrates that a signiﬁ- cant natural source of sulfate exists in some clouds catego- rized as marine and highly marine. In fact, nss-SO2− 4 over the oceans derives principally from reduced sulfur gases, primar- ily dimethylsulﬁde (DMS), which is produced by biological activity in the oceans and from sulfur compounds, primarily SO2, derived from anthropogenic sources on the continents (Savoie et al., 2002). Sulfate can also be produced in cloud water by oxidation of SO2 (Ghauri et al., 2001), which tends to acidify cloud droplets and lead to the production of new aerosol mass by nonvolatile sulfate. In this study, 17 cloud samples (i.e., 7 events) are classi- ﬁed as polluted clouds in comparison with the 52, 101 and 25 samples for continental, marine and highly marine air masses respectively. From Fig. 4, we can conclude that the relative distribution of inorganic ions does not differ strongly for continental and marine air masses, even if the concentra- tions of the quantiﬁed species were different (see Table S2). This observation is well illustrated by the radar chart pre- www.atmos-chem-phys.net/14/1485/2014/ Atmos. Chem. Phys., 14, 1485–1506, 2014 L. Deguillaume et al.: Classiﬁcation of clouds sampled at the puy de Dôme (France) p The amount of carbonyls in cloud water is higher for pol- luted and continental clouds, reﬂecting the great variety of their possible primary (vehicle exhaust, biomass burning, in- dustrial and plants emissions) and secondary (oxidation of hydrocarbons) sources (Altshuller, 1993; Hoekman, 1992; Possanzini et al., 2002). As expected, formaldehyde is the most abundant one in all cases, with concentrations rang- ing from 5.0 µM (for polluted clouds) to 1.8 µM (for marine ones). It is noteworthy that, except in marine samples, HA concentrations are higher than those of GL and subsequently those of MGL. Even if clouds form in rather different atmo- spheric environments, this result supports the observations by Matsunaga and Kawamura (2000) that HA was the main α-oxygenated carbonyl in snow and rain samples. These au- thors attributed the relatively high abundance of HA in their samples to the fact that this carbonyl compound could form a cyclic hemiacetal dimer much less reactive toward oxidation than the free HA molecule. Despite the relatively high gas- phase concentrations expected for this compound (> 1 ppbv) (Matsumoto et al., 2005), concentrations of AC are lower compared to other carbonyls. This can be explained by its low Henry’s law constant (11.4 M atm−1, while the other Henry’s law constant ranged from 2.97 × 103 M atm−1 for F to more than 3 × 105 M atm−1 for GL; Sander et al., 2006). These compounds are not well correlated, demonstrating the multiplicity of sources existing in the atmosphere and the dif- ferent chemical transformations they can undergo once they are dissolved into cloud water. Only two cloud samples considered here are in the pol- luted category. Even though the amount of carboxylic acids was different for each category (see Table S2), the relative distributions for continental, marine and highly marine cat- egories are similar and differ strongly from the polluted air masses, as illustrated by the radar chart presented in Fig. 6. Atmos. Chem. Phys., 14, 1485–1506, 2014 www.atmos-chem-phys.net/14/1485/2014/ L. Deguillaume et al.: Classiﬁcation of clouds sampled at the puy de Dôme (France) Carbonyl compounds have also been monitored at the pdD station: formaldehyde (F) was measured since 2000, and ac- etaldehyde (AC), glyoxal (GL), methylglyoxal (MGL) and hydroxyacetaldehyde (HA) were also quantiﬁed for 24 cloud samples between 2008 and 2011. Carbonyls in cloud water essentially result from their dissolution from the gas phase into the aqueous phase according to their Henry’s law con- stants (Matsumoto et al., 2005). In the aqueous phase, the oxidation of carbonyls produces carboxylic acids (Legrand et al., 2007) and also potentially leads to the formation of oligomers (Ervens and Volkamer, 2010) which can con- tribute, upon cloud droplets’ evaporation, to SOA. Particu- larly interesting, with this scope in mind, is the key role of α-oxygenated carbonyls GL, MGL and HA measured in this study. Oxidation by the HO q radical of these highly water- soluble compounds (Henry ’s law constant HGL = 3.6 × 105; Concentrations of carbonyls in cloud water represent only a small fraction of the organic matter in cloud water (from 0.8 to 1.8 % of the TOC depending on the air mass origins). However, quantifying their concentrations in other cloud samples at various sites would help for further investigations dedicated to the understanding of the in-cloud processes in- volved in the formation of SOA (Ervens et al., 2011, 2013; Tan et al., 2010). In particular, the importance of measur- ing HA when analyzing cloud water chemical composition should be highlighted due to HA’s relatively high concentra- tion. Atmos. Chem. Phys., 14, 1485–1506, 2014 www.atmos-chem-phys.net/14/1485/2014/ 1496 L. Deguillaume et al.: Classiﬁcation of clouds sampled at the puy de Dôme (France) Distribution of carboxylic acids (formic, acetic, oxalic, malonic and succinic acids) for each air mass category (percentages ed using concentration of individual ion in µeq L−1). On the left-hand side, the sum of the concentrations of these compound 1 is indicated as well as the percentage obtained by comparison with the TOC. The radar chart shows the relative contribution of e ion as a function of the air mass categories. Fig. 6. Distribution of carboxylic acids (formic, acetic, oxalic, malonic and succinic acids) for each air mass category (percentages are calculated using concentration of individual ion in µeq L−1). On the left-hand side, the sum of the concentrations of these compounds in µgC L−1 is indicated as well as the percentage obtained by comparison with the TOC. L. Deguillaume et al.: Classiﬁcation of clouds sampled at the puy de Dôme (France) The radar chart shows the relative contribution of each organic ion as a function of the air mass categories. L. Deguillaume et al.: Classiﬁcation of clouds sampled at the puy de Dôme (France) Fig. 7. Distribution of carbonyls (formaldehyde, acetaldehyde, glyoxal, methylglyoxal, hydroxyacetaldehyde) as a function of air categories (percentages are calculated using concentration in µM). On the left-hand side, the sum of the concentrations of these comp in µgC L−1 is indicated as well as the percentage obtained by comparison with the TOC. The radar chart shows the relative contribut each compound as a function of the air mass category. Fig. 7. Distribution of carbonyls (formaldehyde, acetaldehyde, glyoxal, methylglyoxal, hydroxyacetaldehyde) as a function of air mass categories (percentages are calculated using concentration in µM). On the left-hand side, the sum of the concentrations of these compounds in µgC L−1 is indicated as well as the percentage obtained by comparison with the TOC. The radar chart shows the relative contribution of each compound as a function of the air mass category. where a signiﬁcant anthropogenic component is added to the background level (Majestic et al., 2009). near sources of heavy pollution, such as big cities or indus- trial areas (Kotronarou and Sigg, 1993); but they are in the same range as ﬁeld measurements carried out at free tropo- spheric and marine sites such as southern Germany (Sinner et al., 1994), Whiteface Mountain in the US (Arakaki and Faust, 1998), Great Dun Fell in the UK (Sedlak et al., 1997) or San Pedro Hill in the USA (Erel et al., 1993). However, the iron concentrations are signiﬁcantly higher in polluted air masses Figure 8 shows the aqueous concentrations of soluble iron (Fe) in its two main oxidation states: Fe(II) and Fe(III). The ratio between the concentrations of Fe(II) and Fe(III) is a good indicator of the oxidizing capacity of hydrometeors. At the pdD station, Fe(II) is the predominant oxidation state of iron in the cloud water sampled: 83, 70, 54, and 77 %, re- spectively, for the polluted, continental, marine, and highly www.atmos-chem-phys.net/14/1485/2014/ 3.5 Oxidants: iron and hydrogen peroxide to drive the oxidative capacity of the cloud water through the production of HO q radicals (Long et al., 2013) but also accel- erate the conversion of sulﬁte to sulfate (Harris et al., 2013). Measurements of iron concentrations in its different oxida- tion states were conducted on 129 samples (Table S1). The range of concentrations measured at the pdD exhibits much lower values than measurements in clouds or fogs collected Transition metal ions (TMI) are highly reactive in aqueous solutions and their reactivity is dependent on many factors (such as pH), so that their oxidation state and their concen- trations are still prone to large uncertainties (Deguillaume et al., 2005). These compounds, especially iron, are supposed www.atmos-chem-phys.net/14/1485/2014/ Atmos. Chem. Phys., 14, 1485–1506, 2014 1497 L. Deguillaume et al.: Classiﬁcation of clouds sampled at the puy de Dôme (France) 4 Conclusions and recommendations marine categories. This conﬁrms the previous results ob- tained at the pdD by Parazols et al. (2006) who found an average Fe(II) / Fe(tot) ratio of 0.76 ± 0.25 (Parazols et al., 2006). Siderophores produced by microorganisms or macro- molecules such as HULIS or exopolymeric substances (EPS) could play a role in stabilizing iron in its reduced oxida- tion state and preventing its further re-oxidation. This is sup- ported by the correlation existing between TOC and Fe(II) for continental and marine categories (R = 0.70 and 0.61, re- spectively). Interestingly, no relationship is observed for pol- luted and highly marine air masses. Clouds incorporate the local pollution when droplets nucle- ate and then transform pollutants during their lifetime. In this context, the impact of regional and long-range transport of chemical constituents at the puy de Dôme site is apparent and due to the remote (and elevated) geographical charac- ter. In this paper, a long-term database of the aqueous-phase chemical composition of clouds sampled at the puy de Dôme station between 2001 and 2011 is presented. The abundance of inorganic and organic chemical compounds as well as a series of physicochemical parameters permitted the identi- ﬁcation of various inﬂuences corresponding to distinct air- mass histories. A classiﬁcation was performed by principal component analysis that allows for deﬁning the discriminat- ing factors (pH and concentrations of Na+, Cl−, NO− 3 , NH+ 4 and SO2− 4 ) for four different categories: polluted, continen- tal, marine and highly marine. H2O2, like iron, is a strong oxidant of the cloud wa- ter; it originates from gas-to-liquid partitioning of H2O2 and HO q 2 radicals and from aqueous phase photo-production (Anastasio et al., 1994; Laj et al., 1997a). Measurements of the concentration of H2O2 were performed for 107 sam- ples; it ranged from 0.1 to 57.7 µM (Table S2). Figure 8 shows the concentration of H2O2 for each air mass category. The concentrations of H2O2 are much more varied in ma- rine and highly marine air masses (standard deviation = 7.12 and 6.31 µM for highly marine and marine categories, re- spectively) compared to polluted clouds (standard devia- tion = 1.57 µM) characterized by the lowest average concen- tration of H2O2 (∼4.9 µM). L. Deguillaume et al.: Classiﬁcation of clouds sampled at the puy de Dôme (France) 1498 Table 3. Minimum, maximum and mean values of concentrations (µM) of the 5 measured aldehydes measured for the different categories of cloud events. BDL: below detection limit (the limit of detection (LOD) is 0.1 nM for hydroxyacetaldehyde, acetaldehyde, glyoxal, and methylglyoxal, and 0.8 nM for formaldehyde). Concentrations (µM) Hydroxyacetaldehyde Formaldehyde Acetaldehyde Glyoxal Methylglyoxal Polluted Av 1.24 4.97 0.42 0.89 0.19 Min 0.44 1.75 0.05 0.38 0.07 Max 1.77 9.83 0.96 1.87 0.39 Continental Av 0.79 5.06 0.31 0.73 0.22 Min 0.05 0.73 0.02 0.17 0.01 Max 2.34 9.80 0.64 2.29 1.10 Marine Av 0.13 1.76 0.23 0.13 0.01 Min 0.02 0.13 0.05 0.01 BDL Max 0.30 14.25 0.44 0.74 0.03 Highly marine Av 0.64 2.01 0.52 0.23 0.02 Min 0.28 0.70 BDL 0.01 BDL Max 1.24 4.20 1.00 0.36 0.06 www.atmos-chem-phys.net/14/1485/2014/ Atmos. Chem. Phys., 14, 1485–1506, 2014 www.atmos-chem-phys.net/14/1485/2014/ L. Deguillaume et al.: Classiﬁcation of clouds sampled at the puy de Dôme (France) 1499 present in its oxidation state (+II) in all categories of air masses. This potentially indicates that iron could be stabi- lized in its reduced oxidation state by the presence of strong complexing agents. Finally, H2O2 concentrations were much more varied in marine and highly marine clouds than in pol- luted clouds, which were characterized by the lowest average concentration of H2O2. Fig. 8. Distribution of Fe(II), Fe(III) and H2O2 as a function of air mass categories. Numbers above each box plots represent the num- ber of samples analyzed. At least 3 data are required to represent a box plot. The bottom and top of box plots are 25th and 75th per- centiles, respectively. The full line and the square symbol represent the median and mean values, respectively. The ends of whiskers are 10th and 90th percentiles. Statistical differences (Kruskal–Wallis test; p value < 0.05) between groups are indicated by symbols above box plots. As shown in this study, chemical speciation is a powerful tool for analyzing air mass history. For example, the pres- ence of inorganic compounds such as nitrate and sulfate in- vestigated in this study are due to anthropogenic emissions, such as those from automobiles or power production. How- ever, it is difﬁcult to differentiate the various sources of these measured chemicals in cloud water due to the various po- tential processes (transfer from the gas phase, dissolution of particles, aqueous chemical reactivity). In this context, some chemical compounds that are molecular markers of emis- sions (such as levoglucosan, a marker of biomass burning) should be followed in the future in order to correlate cloud features with emissions. Our results also emphasize that, although this database is rather complete, the characterization of the organic matter is still incomplete, since around 10 % of the dissolved organic matter is characterized. The measurements of carbonyls in the cloud water should be also continued in the future since they are precursors of oligomers that are observed in sec- ondary organic aerosols (SOA). Polycyclic aromatic hydro- carbons (PAH) (Ehrenhauser et al., 2012), nitrosamines, phe- nols and nitrophenols (Hutchings et al., 2010), volatile aro- matic compounds such as BTEX (i.e., benzene, toluene, ethylbenzene, and xylene) (Hutchings et al., 2009), and pes- ticides (Millet et al., 1997) should be quantiﬁed for health reasons and their photosensitivity in clouds should be stud- ied in natural and under controlled (i.e., in the laboratory) conditions. L. Deguillaume et al.: Classiﬁcation of clouds sampled at the puy de Dôme (France) Important constituents of ambient atmospheric aerosols are also saccharides (Linuma et al., 2009) such as sugar alcohols mono-, di- and trisaccharides, anhydrosug- ars, and polysaccharides; they are released into the atmo- sphere by microorganisms, plants, and animals. For example, levoglucosan derives from cellulose; galactosans and man- nosans derive from hemicelluloses during biomass burning (Medeiros et al., 2006; Schmidl et al., 2008). The intensity and the speciation of the sugar release into the atmosphere are dependent on the season (Fu et al., 2012), and vary with the diurnal cycle (Kundu et al., 2010). Until now, no quanti- tative measurements of sugar concentrations in cloud water have been performed. However, the presence of sugars in this aqueous compartment is likely due to their high solubility in water; their amount in cloud water should be evaluated. Fig. 8. Distribution of Fe(II), Fe(III) and H2O2 as a function of air mass categories. Numbers above each box plots represent the num- ber of samples analyzed. At least 3 data are required to represent a box plot. The bottom and top of box plots are 25th and 75th per- centiles, respectively. The full line and the square symbol represent the median and mean values, respectively. The ends of whiskers are 10th and 90th percentiles. Statistical differences (Kruskal–Wallis test; p value < 0.05) between groups are indicated by symbols above box plots. The oxidizing capacity of the cloud aqueous phase should also be quantiﬁed for various cloud events. This can be done by the quantiﬁcation of the photo-production of hydroxyl radicals (HO q) as a function of cloud chemical composi- tion, allowing for the identiﬁcation of the different sources and sinks of the most important oxidant of the atmosphere. less abundant in all categories of air masses. Carbonyl com- pounds were less abundant than carboxylic acids with a pre- dominance of formaldehyde and unexpected high concentra- tions of hydroxyacetaldehyde. Iron concentrations were sig- niﬁcantly higher for polluted air masses, and iron was mainly www.atmos-chem-phys.net/14/1485/2014/ Atmos. Chem. Phys., 14, 1485–1506, 2014 4 Conclusions and recommendations Potential explanations are re- lated to (1) efﬁcient sinks of gaseous H2O2 by its reactions with NOx and SO2 present in large concentration and (2) to the efﬁcient reduction of H2O2 in the aqueous phase by its reaction with sulﬁte leading to the sulfate production (Bene- dict et al., 2012; Shen et al., 2012). In addition, the study of Marinoni et al. (2011) presents a diurnal trend with higher day-time concentration of aqueous H2O2, and concludes that the dominant source of hydrogen peroxide is the mass trans- fer from the gas to the liquid phase (Marinoni et al., 2011). As can be expected, highly marine clouds exhibited high concentrations of Na+ and Cl−; the marine category pre- sented lower concentrations of ions but still elevated pH. Fi- nally, the two remaining clusters were classiﬁed as “conti- nental” and “polluted”; these clusters had the second-highest and highest levels of NH+ 4 , NO− 3 , and SO4− 2 , respectively. Furthermore, it was found that total organic carbon (TOC) is signiﬁcantly higher in polluted air masses than in the other categories, due to additional anthropogenic sources. Concen- trations of carboxylic acids and carbonyls represented around 10 % of the organic matter in all categories of air masses. Acetic and formic acids were the most abundant of the ﬁve measured carboxylic acids, reﬂecting their multiple sources (gas phase, particle, aqueous reactivity) in the cloud aque- ous phase. Oxalic acid, succinic and malonic acids were Atmos. Chem. Phys., 14, 1485–1506, 2014 www.atmos-chem-phys.net/14/1485/2014/ L. Deguillaume et al.: Classiﬁcation of clouds sampled at the puy de Dôme (France) L. Deguillaume et al.: Classiﬁcation of clouds sampled at the puy de Dôme (France) Atmos. Chem. Phys., 14, 1485–1506, 2014 Supplementary material related to this article is available online at http://www.atmos-chem-phys.net/14/ 1485/2014/acp-14-1485-2014-supplement.pdf. Arakaki, T. and Faust, B. C.: Sources, sinks, and mechanisms of hydroxyl radical (OH) photoproduction and consumption in authentic acidic continental cloud waters from White- face Mountain, New York: The role of the Fe(r) (r = II, III) photochemical cycle, J. Geophys. Res., 103, 3487–3504, doi:10.1029/97jd02795, 1998. Asmi, A., Wiedensohler, A., Laj, P., Fjaeraa, A.-M., Sellegri, K., Birmili, W., Weingartner, E., Baltensperger, U., Zdimal, V., Zikova, N., Putaud, J.-P., Marinoni, A., Tunved, P., Hansson, H.- C., Fiebig, M., Kivekäs, N., Lihavainen, H., Asmi, E., Ulevicius, V., Aalto, P. P., Swietlicki, E., Kristensson, A., Mihalopoulos, N., Kalivitis, N., Kalapov, I., Kiss, G., de Leeuw, G., Henzing, B., Harrison, R. M., Beddows, D., O’Dowd, C., Jennings, S. G., Flentje, H., Weinhold, K., Meinhardt, F., Ries, L., and Kul- mala, M.: Number size distributions and seasonality of submi- cron particles in Europe 2008–2009, Atmos. Chem. Phys., 11, 5505–5538, doi:10.5194/acp-11-5505-2011, 2011. Acknowledgements. We would like to thank the Regional Council of Auvergne for PhD grants provided to T. Charbouillot, M. Joly and M. Parazols. M. Vaïtilingom acknowledges PhD scholarships from the French Ministry of Research. This work on the long-term analysis of the cloud water chemical composition was supported by the French Ministry and CNRS-INSU through the ORE/SO-BEAM project. Authors acknowledge additional ﬁnancial support from the Regional Council of Auvergne, from the Observatoire de Physique du globe de Clermont-Ferrand (OPGC) and from the Fédération de Recherche en Environnement. The authors also thank C. Bernard for his technical support regarding the cloud sampling. V., Aalto, P. 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https://openalex.org/W2803422577	http://pdf.blucher.com.br/designproceedings/cid2017/26.pdf	Portuguese	null	Design e narrativas: Reflexões contemporâneas	Blucher Design Proceedings	2,018	cc-by	6,811	Design e narrativas: Reflexões contemporâneas Marcos Maia; palavras-chave: Design; Narrativas; Reflexões; Mito; Comunicação Design; Narrativas; Reflexões; Mito; Comunicação 1. Design e narrativas: Reflexões contemporâneas Quais são as possíveis intersecções entre os estudos narrativos e os estudos de design? Podemos pensar no design da narrativa, ou nas narrativas do Design? Que outras abordagens são possíveis quando investigamos apontamentos teóricos que permitem essa aproximação? Para Sudjic (2010, p.34) o design não se baseia mais em modelos mecânicos. Para o autor, resta ao designer “a superfície, a aparência e as nuances semânticas do significado que nos permitem interpretar o que um objeto está tentando nos dizer sobre si mesmo [...] transformam o designer em um narrador". O autor faz a primeira aproximação já estabelecendo vários pontos que podemos investigar: “semântica do significado” “objeto fala sobre si” e “designer como narrador”. O autor completa atribuindo a responsabilidade da eficácia a uma boa história, quando diz que “sem dúvida seja verdade que o design é uma linguagem, só quem tem uma história convincente para contar sabe como usar essa linguagem de maneira fluente e eficaz”. Antes de aproximarmos Design e Narrativas precisamos estabelecer, então, essas categorias ou estruturas que permitam essa aproximação. Partindo das pistas deixadas por Sudjic (2010) das possíveis relações do design com a narrativa, consideramos a história do conceito de narrativas, onde encontramos referência aos enunciados narrativos e suas preposições, categorias de interesse dos estudos de linguagem. Narrativas são desenvolvidas por sujeitos num contexto comunicacional. Adam (1985, p.37) define a proposição narrativa como uma combinação de uma ou mais funções com um ou mais atores. “Uma proposição narrativa se apresenta como um predicado relacionado a ‘n’ argumentos-papéis narrativos”. Utilizando como substrato a teoria semiótica, Romanini (2008, p. 3) também trata o produto do trabalho do designer como uma proposição, quando diz que: “no processo interpretativo, visto sob o prisma do usuário, é o produto criado pelo designer que se posiciona como signo-mensagem capaz de representar o conceito incorporado no produto e gerar, nesse usuário, um efeito. É nesse sentido que um produto de design equivale a uma proposição” (ROMANINI, 2008). Já temos um primeiro indício da aproximação do design das categorias trabalhadas na narrativa. Para entender possíveis relações do estado da arte do Design e das Narrativas precisamos entender a conceituação de Design contemporaneamente. 1. Design e narrativas: Reflexões contemporâneas Diversos outros autores nos permitem pensar o design como linguagem conforme explicam, utilizando como entendimento para essa relação sua manifestação como produção cultural, além de atentarem para utilidade dos “estudos das linguagens para o campo do design, principalmente quando se trata dos seus aspectos comunicativos e de produção de significados” (BRAIDA e NOJIMA, 2016, p.43). resumo: O presente artigo faz reflexões acerca das relações entre o design e as narrativas, buscando encontrar pontos de similaridade metodológica ou conceitual nos vários autores aqui citados. Nossa proposta é propor reflexões a partir da aproximação do design por um viés contemporâneo das teorias e práticas narrativas, percorrendo por conceitos e teorias como semiótica, narratologia, mito, consumo, psicologia, realidades narrativizadas, design interativo, ficção especulativa, design de narrativas, design estratégico, visando trazer alguns caminhos que possibilitam novas pesquisas e reflexões acerca do design. Tendo em vista a noção de design enquanto linguagem, podemos tentar abordar o potencial narrativo do design e diferentes campos do estudo que fazem essa aproximação, Embora o pensamento cartesiano tenha influenciado nosso pensar e fazer, ainda somos seres que se comunica por meio de histórias, desde o pensamento mitológico que visava explicar os fenômenos do mundo e a nós mesmos até os dias de hoje, evoluindo suas estratégias, perpassando as organizações sociais e políticas, as formas de comunicação e utilizando a evolução tecnológica, emergindo entre variados suportes até as narrativas transmidiáticas. De única forma de verdade e percepção do real, as formas narrativas foram descreditadas frente ao pensamento científico, e o mito virou sinônimo de mentira, passando novamente a ser consideradas as portadores dos aspectos culturais e hoje podemos rever a utilização dos elementos narrativos como forma de alteração da realidade. É no potencial das narrativas, de mobilizarem a crença, que reside a força das narrativas, onde o pensamento científico não alcança com a mesma capacidade de adesão. Tal como a verbal e imagética, o design é linguagem que materializa signos e conceitos em artefatos, e como as demais formas de comunicação poderia utilizar-se dos elementos narrativos para construir seu discurso aproveitando as possibilidades que trazem as narrativas que perpassam e constroem o mundo. Somos uma espécie movida por histórias e esse aspecto estruturante da nossa cognição pedem um esforço interdisciplinar para a construção de uma metodologia e epistemologia ainda pouco desenvolvida no design. 1.1 Design como comunicação O trabalho do designer é análogo ao do comunicador (ROMANINI, 2008, p.3). O autor atenta para a visão de que uma proposição só possa assumir a forma de símbolos, como palavras de uma língua. Essa visão excluiria o Design do escopo dos estudos comunicacionais. O conceito é o tradicional de proposição. Quando surge a semiótica de Peirce, esses limites são ampliados, pois a semiótica traz a ideia de que muito mais do que “conceitos travestidos de símbolos”. Para o autor, o produto do design também guarda sempre uma gama de possibilidades interpretativas por parte do intérprete, que pode, inclusive, ressignificar com alguma liberdade, podendo obter alguns novos usos e interpretações, inclusive gerando novas proposições. Desta forma, poderíamos perceber o designer como um emissor, no processo comunicativo, fazendo uma sintaxe entre a forma que elaborou e a materialidade que tem à sua disposição para então comunicar suas ideias, produzindo proposições que podem criar efeitos na mente do intérprete- receptor (ROMANINI, 2008). Se percebermos os designers como produtores de proposições, para além das definições de solucionador de um problema específico, podemos ampliar as possibilidades de criação do designer enquanto comunicador, muito além da mobilização de significados para atender as necessidades de utilidade, ou mesmo de geração de valor, é possível transmitir muito mais informações. enquanto comunicador, muito além da mobilização de significados para atender as necessidades de utilidade, ou mesmo de geração de valor, é possível transmitir muito mais informações. Cardoso (2013, p.141) considera que “o que importa, em termos de design, é que a capacidade das formas de comunicar informações à mente humana é muito mais profunda e abrangente do que 'simplesmente' o conjunto de significados impostos pela sequência fabricação, distribuição e consumo”. Se falamos em intérprete-receptor, não podemos esquecer que ainda se trata de um consumidor. O próprio conceito de consumo é contemporâneo e alinhavado ao conceito que estamos utilizando para a atribuição de significados aos produtos do design. Pois não são os objetos materiais que são consumidos, pois são apenas objeto de necessidade e de satisfação. “Para tornar-se objeto de consumo o objeto precisa se tornar um signo” (BAUDRILLARD, 2006, p.207). O autor explica que: “exterior de alguma forma a uma relação da qual apenas significa - 'portanto arbitrário' e não coerente com esta relação concreta, mas adquirindo coerência e consequentemente sentido em uma relação abstrata e sistemática com todos os outros objetos-signos. 1.1 Design como comunicação É então que ele se "personaliza", que entra na série, etc.: é consumido - jamais na sua materialidade mas na sua diferença (BAUDRILLARD, 2006, p.207). “exterior de alguma forma a uma relação da qual apenas significa - 'portanto arbitrário' e não coerente com esta relação concreta, mas adquirindo coerência e consequentemente sentido em uma relação abstrata e sistemática com todos os outros objetos-signos. É então que ele se "personaliza", que entra na série, etc.: é consumido - jamais na sua materialidade mas na sua diferença (BAUDRILLARD, 2006, p.207). Para Baudrillard (2006), o consumo não se trata de uma prática material, portanto, não se trata da alimentação, vestuário, posse de veículos, ou mesmo a substância (verbal e não verbal) das imagens e mensagens , mas por tudo enquanto substância significante. O consumo seria, então, a “totalidade virtual” de todos os objetos e mensagens constituídos em um discurso cada vez mais coerente. Como se trata de ter coerência, ou seja, possuir um sentido, se trata de “uma atividade de manipulação de signos” (BAUDRILLARD, p. 206). Essa manipulação pode ser positiva ou negativa, mas ela pode ser exercita pelo designer, assim como pelo comunicador. Conforme Sudjic (2010, p.49) o design "pode ser usado de formas manipuladoras e mal-intencionadas, ou criativas e ponderadas. O design é a linguagem que ajuda a definir, ou talvez a sinalizar, valor”. É uma linguagem que a sociedade se vale para criar objetos que reflitam seus valores e objetivos. Esta mesma linguagem do design também será utilizada para sugerir diversas características do próprio objeto como o gênero, podendo ser com uso da cor, forma, tamanho ou referências visuais diversas, pois, conforme Sudjic (2010, p.21) “o design passou a ser a linguagem com que se molda esses objetos e confecciona as mensagens que eles carregam”. O autor explica que "o design é usado para moldar percepções de como os objetos devem ser compreendidos. As vezes, é uma questão de comunicação direta [...] às vezes, a comunicação é mais emocional" (SUDJIC, 2010, p.51). BRAIDA e NOJIMA (2016) lembram de alguns autores como Burdek (2006, p.283) que atenta para os primeiros estudos a considerar o design como fenômeno de linguagem, considera sua função comunicativa, e Moura (2003, p.102) que afirma ser o design uma linguagem, pois design está inserido na cultura, tal como atua com ela. 1.2 O design do mito No capítulo sobre os objetos antigos, Baudrillard (2006) fala do fenômeno do interesse pelos objetos antigos e do valor simbólico dos objetos antigos. Diz que para o homem se sentir em casa, precisa utilizar objetos antigos como reorganizadores do mundo, o que o objeto funcional não consegue fazer. Enquanto o objeto funcional se esgota na cotidianidade, o objeto antigo surge como uma dimensão regressiva que testemunha um fracasso relativo do sistema, mas paradoxalmente, o faz funcionar. A ambiguidade deve-se à densidade inconsciente do objeto antigo que atua como um talismã que guarda a sabedoria dos anciãos. Ao passo que objetos não dependem da ambiência, não serve pra nada, serve profundamente para qualquer outro. "de onde surge essa motivação tenaz para o antigo, o velho móvel, o autêntico, o objeto de estilo, o rústico, o artesanal, o feito à mão, a cerâmica indígena, o folclore etc.? De onde surge esta espécie de fenômeno de aculturação que arrasta os civilizados para os signos excêntricos, no tempo e no espaço, do seu próprio sistema cultural, para os signos que arrasta os subdesenvolvidos para os produtos industriais?" (BAUDRILLARD, 2006, p.83). A exigência à qual os objetos antigos respondem é de ser um ser definitivo. Para Baudrillard (2006, p.83) "o tempo do objeto mitológico é o perfeito" pois ele ocorre no presente como se tivesse ocorrido outrora por este motivo acha-se fundado sobre si, "autêntico". O objeto antigo é sempre, no sentido exato do termo, um "retrato de família". Sob a forma concreta do objeto a imemorização de um ser precedente existe - processo equivale, na ordem imaginária, a uma elisão do tempo. É o que falta aos objetos funcionais, que existem somente na atualidade, se esgotando em seu próprio uso, se assegurando no espaço, mas não no tempo. Baudrillard (2006) vai responder sua própria pergunta do porquê do interesse pelos objetos antigos, dizendo que "o objeto funcional é eficaz, o mitológico, perfeito". O autor completa que “O objeto antigo dá-se, portanto como mito de origem”. j g p g Quando falamos em mito, estamos falando na historicidade do objeto, isto é, uma narrativa que o objeto foi adquirindo com o passar do tempo, sua própria história no percurso do tempo. Falar em mito é falar em narrativa. Narrar é uma ação que acompanha a humanidade há séculos, desde o surgimento do pensamento mítico visando explicar o próprio homem, o mundo e seus fenômenos. 1.1 Design como comunicação Essas ideias vêm sendo trabalhadas pelos teóricos do design desde a década de 70, e am sendo aprimoradas. Para Sudjic (2010, p.51) o design “é uma linguagem que evolui e modifica seus significados tão depressa como qualquer outra. Pode ser manipulada com sutileza e inteligência, ou com obviedade canhestra. Mas a chave para entender o mundo feito pelo homem”. Para Sudjic (2010, p.51) o design “é uma linguagem que evolui e modifica seus significados tão depressa como qualquer outra. Pode ser manipulada com sutileza e inteligência, ou com obviedade canhestra. Mas a chave para entender o mundo feito pelo homem”. Para o autor, o papel dos designers mais sofisticados, hoje, tanto é ser contadores de histórias, fazer um design que fale de uma forma que transmita essas mensagens, quanto resolver problemas formais e funcionais. (SUDJIC, 2010, p..21) 1.2 O design do mito A representação temporal dos eventos ainda é um elemento-chave para a compreensão da narrativa, mesmo que essa ordem não precise seguir uma sequência inflexível. A não cronologia da ordem não destitui o caráter narrativo. O autor Ricoeur (1994) vai considerar a narrativa como a representação mais clara e exata do tempo, como um caráter temporal da existência do homem. Para tal, parte das ideias de Santo Agostinho (1987) que afirma só se poder definir e medir o tempo a partir do interior do homem, pois o pontífice considera a natureza do tempo como sendo psicológica. Jung (1984) em seus estudos também chegou a percepção de uma “estrutura no enunciado narrativo” que vai chamar de drama, remontando à Aristóteles (1922) como outros autores já que Aristóteles continua sendo uma referência para os estudos de narrativas, que ele vai chamar de drama. Para Jung (1984) estrutura narrativa se organiza com quatro macroproposições: exposição, desenvolvimento, peripécia e resultado, obtidas a partir da análise dos sonhos. Os sonhos são fonte profícua para obter respostas sobre nossa própria origem e nosso futuro. Sua relação com o pensamento mitológico é inegável e relacionado aos arquétipos que nos remetem ao pensamento mítico. As religiões, filosofias, artes, formas sociais do homem primitivo e histórico, descobertas fundamentais da ciência e da tecnologia e os próprios sonhos que nos povoam o sono surgem do círculo básico e mágico do mito. As religiões, filosofias, artes, formas sociais do homem primitivo e histórico, descobertas fundamentais da ciência e da tecnologia e os próprios sonhos que nos povoam o sono surgem do círculo básico e mágico do mito. O prodígio reside no fato de a eficácia característica, no sentido de tocar e inspirar profundos centros criativos, estar manifesta no mais despretensioso conto de fadas narrado para fazer a criança dormir — da mesma forma como o sabor do oceano se manifesta numa gota ou todo o mistério da vida num ovo de pulga. Pois os símbolos da mitologia não são fabricados; não podem ser ordenados, inventados ou permanentemente suprimidos. Esses símbolos são produções espontâneas da psique e cada um deles traz em si, intacto, o poder criador de sua fonte. Qual o segredo dessa visão intemporal? De que camada profunda vem ela? Por que é a mitologia, em todos os lugares, a mesma, sob a variedade dos costumes? E o que ensina essa visão? 1.2 O design do mito Temos os registros arqueológicos materiais com arte rupestre que contam-nos narrativas de outra era, e hoje, “as histórias de fadas, contos, piadas, novelas, cinema e tantos outros, passando também pelos mitos e livros sagrados — todos expressam e são alcançados pelas narrativas em vários suportes” (GANCHO, 2006). Conforme explica Hoad (2003), o conceito de narrar, remete ao termo narrare, derivado, por sua vez, do termo latino gnarus, que significa “saber”, “conhecer algo” (a palavra oposta, ignarus, com a adição do prefixo i, formou o adjetivo ‘ignorante’). Teixeira (2015) explica que o termo denota a partilha de conhecimento, o contar e dizer, de modo que se pode entender que narrativa trata-se do próprio ato de contar uma história, enquanto a narração se refere ao conteúdo deste ato. Uma história é um agrupamento de situações narradas, enquanto que a forma como essa história é narrada chama-se enredo. A narrativa é, portanto, composta por enredo, história e narração, conforme Nogueira (2010). Barthes (1975) nos lembra que as formas de narração existentes são diversas e imensuráveis, pois há uma variedade de gêneros que se distribuem por tantas mídias quanto possíveis, sugerindo que todas as substâncias podem ser “invocadas” para dar conta das histórias (BARTHES, 1975, p. 237). O estudo da narrativa tem na obra “Poética” de Aristóteles (1992), uma base de referência que foi escrita em meados de 335 a.C. e continua válida até hoje devido à profundidade de sua análise. Posteriormente Propp (1983) propõe-se a analisar a morfologia dos contos de fada, descrevendo os contos por suas partes constitutivas relacionando-as entre si. O autor Bremond (1966) faz uma revisão dos estudos elaborador por Propp, expandindo-os para além dos contos de fada, tratando das narrativas no geral e elabora um modelo de “estrutura dos enunciados narrativos”. Visando estabelecer uma estrutural fundamental, Greimas (1973), sistematiza quatro etapas formadoras da narrativa: o contrato, a competência, a performance e a sanção, formando um ciclo que para ele, são os elementos que compõem a narrativa. Podemos perceber que temos o estabelecimento de algumas bases como: considerando o enunciado que atende alguns pressupostos como narrativo, por uma visão estruturalista. Para uma narrativa coerente é necessário que as proposições estejam relacionadas de forma cronológica e lógica. Já Goodman (1981), atenta que a simples ordenação de eventos em categorias estéticas ou científicas transforma uma estória em estudo, destituindo seu caráter narrativo. 1.2 O design do mito (CAMPBELL, 1995) Conforme Jung (2000, p.9), os arquétipos “não são disseminados apenas pela tradição, idioma ou migração. Eles podem reaparecer espontaneamente a qualquer hora, em qualquer lugar, e sem qualquer influência externa”. Os arquétipos são modelos universais, predisposições e tendência a experimentar as coisas de determinadas formas. São importantes elementos da psique humana que podem ser encontrados em todos os povos, civilizações, em qualquer era, independente do tempo. O pensamento mítico é a primeira forma que os homens desenvolvem para responder às suas questões existenciais. São narrativas de deuses, heróis e jornadas para ilustrar e explicar os diversos fenômenos, o mundo e o próprio comportamento humano, além de transmitir valores e a própria cultura. Os mitos surgem com o pensamento mítico e são formas de expressão dos arquétipos. Por ser parte de uma tradição cultural, o mito configura assim a própria visão de mundo dos indivíduos, a sua maneira mesmo de vivência esta realidade. Nesse sentido, o pensamento mítico pressupõe a adesão, aceitação dos individuas, na medida em que constitui as formas de sua experiência do real (MARCONDES, 2002, p20). Com o surgimento da filosofia que busca uma forma lógica de pensar, e posteriormente a ciência, mito torna-se um conceito pejorativo, usado para expressar mentira. No entanto, as narrativas seguindo o modelo mítico e usando os arquétipos está presente em diversas áreas da sociedade contemporânea: desde as narrativas jornalísticas aos discursos políticos e das instituições que estruturam e organizam a sociedade da forma que conhecemos. Além de estar artisticamente representada como forma de entretenimento ou forma de transmissão de conhecimento, tanto na educação como na arte, em diferentes formatos e suportes: teatro, cinema, quadrinhos, literatura etc., contando com toda evolução tecnológica. Mentiras disseminadas nas redes sociais podem mobilizar um grupo grande de pessoas e alterar a realidade física a partir das ações de grupo. Os boatos disseminados intencionalmente utilizam dessa força que a forma de contar histórias possui, mobilizando um contingente de pessoas movido mais pelas narrativas que pelos fatos objetivos. Mesmo um filme, série ou novela podem mudar comportamentos e visões acerca de um assunto. 1.2 O design do mito As autoras Ângela Marques e Rousiley Maia (2008, p.165-206) mostram como as telenovelas A Próxima Vítima (Globo, 1995) e Torre de Babel (Globo, 1998/99) que se utilizaram de narrativas de sujeitos homoafetivos que desafiam discursos dominantes da época provocando uma série de argumentações diversas em outros meios de comunicação sobre as questões envolta das pessoas gays e lésbicas: direitos, estilo de vida e demais aspectos relacionados a esta orientação afetivossexual, promovendo a deliberação no espaço público. p p p p Essa tendência narrativa pode ter explicações do campo da psicologia. O autor Bruner (2001) vai além e considera as narrativas um modelo de pensamento. O autor teoriza que a interação do homem com o mundo acontece dialeticamente e para criar essa relação entre “mundo interno” (mente) e “mundo externo” (extracorpóreo, perceptível aos sentidos) utiliza como estratégia o pensamento narrativo e científico. Os dois modos de funcionamento cognitivo constroem realidades, ordenando a experiência cada um da sua forma, construindo realidades. As realidades narrativizadas, eu suspeito, são demasiadamente onipresentes, sua construção é demasiadamente habitual ou automática para ser acessível à fácil inspeção. Vivemos em um mar de histórias, e como os peixes que (de acordo com o provérbio) são os últimos a enxergar a água, temos nossas próprias dificuldades em compreender o que significa nadar em histórias. Não que não tenhamos competência em criar nossos relatos narrativos da realidade longe disso -, somos, isso sim, demasiadamente versados. Nosso problema, ao contrário, é atingir uma consciência do que fazemos facilmente de forma tão automática, o antigo problema da prise de conscience (BRUNER, 2001, p.140). A estrutura do pensamento narrativo concorda com gêneros do discurso internalizados, de forma que é difícil distinguir bem o pensamento do texto narrativo pois o pensamento torna-se inextrincável da linguagem que o expressa. O que nos permite refletir se um produto do design comparado a uma proposição a ser interpretada também pode ser pensado na sua produção tendo em vista o modelo cognitivo narrativo. Joseph Campbell uma das definições mais belas – quase poética – de mitologia: “A mitologia é a canção do universo – música que nós dançamos mesmo quando não somos capazes de reconhecer a melodia” (1990, p. XI). Tal como Bruner, Campbell sugere que fazemos intuitivamente algumas coisas, corroborando para uma visão de um pensamento narrativo, ou seja, mitológico, ainda que Campbell esteja se referindo aos arquétipos. 1.3 Design de ficção A tendência narrativa do homem nos leva a desenvolver diferentes formas de contar histórias. Além de nos apropriarmos das diversas tecnologias para contar essas histórias. Para Sutton (1997) toda nova linguagem é poética e metafórica num primeiro momento. Uma nova imagem permite a possibilidade de significar algo, para poder submetê-la ao juízo e à crítica e à comprovação experimental. Para o autor, toda nova linguagem começa interpretativa tentando comparar e compartilhar interpretações para chegar a acordos sobre como percebemos as coisas (p.18). Sutton (1997) se refere a novos paradigmas para o ensino de ciências. Mas o mesmo ocorre em diversas esferas da vida cotidiana, em que trabalha-se de forma lúdica, isto é, com recursos narrativos para abordar os temas. As novas tecnologias permitem ao homem outros canais e recursos para essa comunicação. Como campo para desenvolvimento de discursos narrativos, além das mídias como jogos, quadrinhos, literatura, teatro e outras que podem ser utilizadas pelo design, podemos destacar o próprio design como canal para esses discursos narrativos quando falamos de design interativo e design especulativo. PAPANEK (1993) considera que “um designer, no sentido mais lato do termo, é um ser humano que percorre com êxito a estreita ponte que liga aquilo que nos foi deixado pelo passado às possibilidades futuras”(p.215). p (p ) O Autor Heinlein (1947) cunhou o termo “ficção especulativa” (reduzido para spec-fi) sugerindo que as ficções não precisariam utilizar somente a ciência como base. Ele sugere que o processo pode ser mais amplo e variar. Para Jones (2015) todo design é ficção até certo ponto, antes de se concretizar. Quando se projeta para um futuro ou mundo novos, entender os detalhes desse mundo em que pensamos, torna-o mais crível. Por conter, nessa elaboração ficcional, especulações e mobilizar símbolos em intenções, o design traz consigo princípios de narratividade. Já Sterling (2009) pondera que o processo criativo pode ser propositivo em relação a inovações tecnológicas, demonstrando que a diégese de obras midiáticas podem se apropriar das metodologias do design. As interfaces do dia a dia possuem semelhanças com as ficções especulativas, pela possibilidade de transformação ou mesmo a ubiquidade da internet das coisas, especialmente a noção diegética do tempo, uma narrativa de cada interação nas interfaces digitais e artefatos. A fragmentação da comunicação nos diversos suportes permitem uma dinâmica assíncrona em que o tempo e lugar do emissor não são tão relevantes, o que Baudrillard (1983) chama de hiper- realidade. 1.2 O design do mito Poderiam ser os arquétipos a forma mais visível no atual estado da arte sobre o estudo das narrativas por esta abordagem. Nicola Abbagnano (2000) agrupa diversos conceitos de autores e chega a três visões do mito: A primeira como contrária ao Lógos, à Razão e ao discurso filosófico, numa visão histórica linear; a segunda entendendo o mito como “uma forma autônoma de pensamento ou de vida” (p.673) podendo ser relacionada às ideias de Bruner (2001); e, por último, considera o mito com a função única de dar continuidade à cultura [...] não apenas ligando-o às sociedades antigas, mas indispensável para qualquer cultura, sugerindo “estudá-lo em relação à função que exerce na sociedade humana” (p.675), permitindo-nos perceber as narrativas como partes constituintes das culturas humanas ao passo que dentre outras funções, às promove e perpetua. permitindo-nos perceber as narrativas como partes constituintes das culturas humanas ao passo que dentre outras funções, às promove e perpetua. 1.4 Design de Narrativas (de jogos) Dentre as diferentes interfaces homem-máquina podemos destacar as simulações virtuais e jogos narrativos como interfaces para experiências lúdicas que servem a tantos propósitos quanto possíveis. Um jogo é um sistema onde os jogadores envolvem-se em um conflito artificial, definido por regras, que implica em um resultado quantificável (SALEN; ZIMMERMAN, 2012, p. 95). De outro lado, o design é uma atividade que configura objetos de uso e sistemas de informação visando materializar uma finalidade ideal Bomfim (1999). Essas duas definições destacam semelhanças enquanto sistemas que podem ser relacionados. Fica claro, se consideramos que o jogo é um projeto que respeita os mesmos pressupostos do design, sendo possível aplicar a mesma lógica para identificar a existência de desdobramentos narrativos em ambos, formando-se então, o design de narrativa Oliveira (2014). Design de narrativa é a criação da história e das mecânicas pelas quais passará o jogo, compreendendo não só a história propriamente dita, mas também como ela será comunicada ao jogador, e também como outras funções do jogo farão o jogador imergir no mundo do jogo (POSEY, 2008, p.871). Dentre os jogos que podem ser facilmente reconhecidos como narrativos, existem duas categorias principais: Os jogos de personagem com construção de um ambiente navegável cada vez mais sofisticado no qual a sensação de imersão e de "presença vicária" (Darley, 2002, p.237), cuja imersão se dá através do avatar do protagonista. A linguagem possui heranças do cinema com uso de narrativas clássicas. Já os jogos de simulação se afastam dessa narrativa clássica propondo uma experiência de controle de um sistema como se o jogador fosse um deus que controla aspectos do mundo criado. No entanto, existe uma tendência à hibridação das duas vertentes culminando num formato narrativo próprio. Simulação é um "instrumento fundamental da significação", faz referência à “constituição de um modelo (hipótese teórica) interpretativo a respeito de uma certa realidade e, em segunda instância, à verificação empírica da funcionalidade e da adequação deste modelo” (BETTETINI,1989, p.71) O design de narrativa é planejar a narrativa do jogo verificar se existe coerência entre a narrativa incorporada e a emergente (Oliveira 2013), o autor atenta que a história deve contextualizar e envolver o jogador, com uso de mecânicas que se relacionam com a história proposta. 1.3 Design de ficção Os espaços virtuais e materiais se misturam criando um espaço de narrativa verossímil. A ficção especulativa é responsável por muitos produtos interativos como por exemplo, no filme Minority Report (2002) em que aparece uma tecnologia de interface gestual, se tornando uma referência do imaginário coletivo, por meio de uma narrativa. Outras obras como Star Trek (1960) que previu ou sugeriu as tecnologias de comunicação móveis como celular, ou 007 Goldfinger (1964) que anteviu o GPS. Tal como na arte, o design tem em sua gênese a histórica necessidade do homem de se manifestar criativamente para solucionar problemas do mundo com inovações tecnológicas. Durante o processo de prototipagem do design, são elaborados cenários fictícios, personas, cria-se mundos virtualmente. A interação narrativa-design, por este ponto de vista é relevante para entendermos que muitas soluções materializadas a partir de ficções antigas, outras possibilidades futuras para o design de interação são possíveis principalmente para as interfaces homem-máquina que ainda não são muito atraentes, precisam ser humanizadas, narrativizadas. Pelas transformações sociais e tecnológicas precisamos entender melhor as relações entre design e ficção. 1.4 Design de Narrativas (de jogos) Uma analogia possível com a função do designer de narrativas é em relação ao roteirista hipermídia devido à necessidade de formação interdisciplinar que precisa realizar as funções de um roteirista audiovisual Gosciola (2010), mas superando a linearidade das mídias narrativas como teatro e audiovisuais, considerando a experiência interativa do usuário. Cardoso (2013), traz o entendimento de que o designer leva o jogador a cumprir um papel específico em um sistema que proporciona recompensas de forma coerente e incorporadas à narrativa, como forma de manter sua motivação. Se esse “pacto” entre o designer e o jogador for selado, a experiência do jogador se caracterizará como uma narrativa significante. Considerações Finais Desde o pensamento mítico, evoluindo para as narrativas transmídias contemporâneas, as narrativas passaram de verdade à mentira e, portadoras da cultura e dos valores humanos, são verdades relativas ao alterar as percepções da realidade. “A capacidade de criar histórias que pudessem gerar uma lógica em torno de um evento, seja ele natural ou sobre alguma moral humana, rendeu ao mito a possibilidade do ser humano de conseguir imaginar o não objeto e crer nele como fato” (MERLEAU-PONTY, 1965). É no potencial das narrativas de mobilizarem a crença que reside a força das narrativas, onde o pensamento científico não alcança com tamanha pregnância. É no potencial das narrativas de mobilizarem a crença que reside a força das narrativas, onde o pensamento científico não alcança com tamanha pregnância. p g Somos uma espécie movida por estórias e essa motivação precisa ser consciente no des p g Somos uma espécie movida por estórias e essa motivação precisa ser consciente no design. "Nosso apetite por estórias é um reflexo da necessidade profunda do ser humano em compreender os padrões do viver, não meramente como um exercício intelectual, mas como uma experiência pessoal e emocional. Nas palavras do dramaturgo Jean Anouilh, a ficção dá à vida sua forma” (MCKEE, 2006, p.25). "Nosso apetite por estórias é um reflexo da necessidade profunda do ser humano em compreender os padrões do viver, não meramente como um exercício intelectual, mas como uma experiência pessoal e emocional. Nas palavras do dramaturgo Jean Anouilh, a ficção dá à vida sua forma” (MCKEE, 2006, p.25). A visão contemporânea do design atenta para sua imaterialidade e sua relação enquanto proposição a ser interpretada, pois design é uma forma de comunicação e como linguagem tal como a verbal ou imagética buscar as possibilidades que trazem as narrativas que inundam e constroem nosso mundo. Enquanto áreas de investigação teórica, o design interativo, o design especulativo e as relações entre design e ficção são profícuas, contendo uma série de novos termos numa taxonomia que vem se alterando conforme avançam os estudos. O design de narrativa encontra nos jogos um maior aporte teórico, mas existe espaço e precisa de mais desenvolvimento dos estudos acerca desta e de outras categorias multimídias. Estudos sobre a historicidade e narratividade dos objetos de design podem ser aprofundados, mas pedem um esforço interdisciplinar para a construção de uma metodologia e epistemologia ainda incipiente. 1.5 Uma narrativa estratégica Os jogos permitem uma identificação mais direta dos efeitos que a simulação ou mesmo os jogos de personagem causam no jogador, “substituindo” a realidade material em que se encontram pela realidade criada, ou melhor, alterando a percepção da própria realidade. Mas toda forma de entretenimento como os quadrinhos, literatura, cinema, TV, teatro, entre outras, mesmo uma história contada verbalmente sem nenhum outro recurso senão a fala, consegue o efeito de transportar o intérprete-receptor para o mundo criado narrativamente, podendo alterar a percepção da realidade, sobrepô-la ou mudar conceitos acerca desta realidade. Os Estudos Culturais com as teorias estéticas da recepção ativa (HALL) falam da capacidade de ressignificação dos objetos de consumo (CANCLINI, 1995). E é nesta possibilidade de permitir a ressignificação (ou alteração de elementos da realidade) que o design como teoria e prática mostra um grande potencial. Cardoso (2013, p.20) atenta a importância dos conceitos imateriais quando diz “Em uma palavra, o ‘imaterial’ passou a ser o fator decisivo em quase todos os domínios, mormente numa área como o design”. Essa transição dos conceitos trabalhados nas narrativas e trazidos para a materialidade nos levam a pensar sobre a transição que fazemos ao consumir uma narrativa de forma interativa com a realidade material. Para Cardoso (2013, p.40) “a possibilidade de realizar transições muito rápidas entre material e imaterial é um dos fenômenos mais marcantes da atualidade” (CARDOSO, 2013, p.40). Não só os significados mobilizados no fazer do designer são capazes de gerar efeitos, mas também a disposição destes, no objeto ou com o objeto do designer geram efeitos de sentido e, portanto as narrativas que suscitam devem ser pensadas. Ainda, o objeto material ou imaterial do designer pode ser trabalhado dentro ou junto com narrativas, ampliando as possibilidades da práxis do design. Design and narratives: Contemporary reflections Abstract: The present article reflects on the relations between design and narratives, seeking to find points of methodological or conceptual similarity in the various authors mentioned here. Our proposal is to propose reflections from the approach of design through a contemporary bias of narrative theories and practices, through concepts and theories such as semiotics, narratology, myth, consumption, psychology, narrativized realities, interactive design, speculative fiction, narrative design, strategic design, aiming to bring some paths that make possible new researches and reflections about the design. Given the notion of design as language, we can try to address the narrative potential of design and different fields of study that make this approach. Although Cartesian thinking has influenced our thinking and doing, we are still beings who communicate through stories, from the mythological thought that aimed to explain the phenomena of the world and ourselves up to the present day, evolving their strategies, crossing the social and political organizations, the forms of communication and using technological evolution, emerging from various supports to the transmedia narratives. From the only form of truth and perception of the real, the narrative forms were skewed in the face of scientific thought, and the myth became a synonym of a lie, once again considered the bearers of cultural aspects and today we can review the use of narrative elements as a form of change of reality. It is in the potential of the narratives, to mobilize the belief, that lies the force of the narratives, where scientific thought does not reach with the same capacity of adhesion. Like verbal and visual, design is a language that materializes signs and concepts in artifacts, and like other forms of communication, one could use the narrative elements to construct their discourse, taking advantage of the possibilities that bring the narratives that permeate and construct the world. We are a species driven by stories and this structuring aspect of our cognition calls for an interdisciplinary effort for the construction of a methodology and epistemology still little developed in the design. Keywords: Design; Narratives; Reflections; Myth; Communication Considerações Finais Como modelo cognitivo, o pensamento narrativo propõe reflexões acerca da forma de pensar e da prática do design que podem sugerir caminhos para uma criação mais ciente dos processos narrativos. Evidente que as aproximações entre Design e Narrativas possuem muitos mais pontos de intersecção que os apresentados neste artigo. Entretanto, muitos caminhos aqui citados permitem vislumbrar o mar de possibilidades (narrativas) do qual fala Bruner (2001) e, cientes do potencial que traz o mito, incorporar aos estudos do design, num contexto mais que contemporâneo, atemporal. BAUDRILLARD, Jean. O sistema dos objetos. [Tradução: Zulmira Ribeiro Tavares]. São Paulo: Perspectiva, 2006 BETTETINE, Gianfranco. Por un establecimiento semio-pragmático del concepto de simulación. Videoculturas de fin de siglo. Madrid: Cátedra, 1989 Referências bibliográficas ABBAGNANO, Nicola. Dicionário de Filosofia. São Paulo: Martins Fontes, 2000 ADAM, J-M. Le texte narratif. Paris: Nathan, 1985 Aristóteteles. Poética (E. de Souza, Trad.). São Paulo: Ars Poética, 1992 BARTHES, Roland; DUISIT, Lionel. An Introduction to the Structural Analysis of Narrative. New Literary History. New York, v. 6, n. 2, 1975 Aristóteteles. Poética (E. de Souza, Trad.). São Paulo: Ars Poética, 1992 BARTHES, Roland; DUISIT, Lionel. 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https://openalex.org/W4237555031	https://figshare.le.ac.uk/articles/journal_contribution/Allele_specific_expression_in_worker_reproduction_genes_in_the_bumblebee_Bombus_terrestris/10142165/1/files/18278231.pdf	English	null	Allele specific expression in worker reproduction genes in the bumblebee <i>Bombus terrestris</i>	null	2,015	cc-by	6,227	ABSTRACT Methylation has previously been associated with allele specific expression in ants. Recently, we found methylation is important in worker reproduction in the bumblebee Bombus terrestris. Here we searched for allele specific expression in twelve genes associated with worker reproduction in bees. We found allele specific expression in Ecdysone 20 monooxygenase and IMP-L2-like. Although we were unable to confirm a genetic or epigenetic cause for this allele specific expression, the expression patterns of the two genes match those predicted for imprinted genes. Subjects Animal Behavior, Entomology, Evolutionary Studies, Genetics, Zoology Keywords Social insect, Hymenoptera, Ecdysone, Epigenetics Subjects Animal Behavior, Entomology, Evolutionary Studies, Genetics, Zoology Keywords Social insect, Hymenoptera, Ecdysone, Epigenetics How to cite this article Amarasinghe et al. (2015), Allele specific expression in worker reproduction genes in the bumblebee Bombus terrestris. PeerJ 3:e1079; DOI 10.7717/peerj.1079 Allele specific expression in worker reproduction genes in the bumblebee Bombus terrestris Harindra E. Amarasinghe1,3, Bradley J. Toghill2,3, Despina Nathanael1 and Eamonn B. Mallon1 1 Department of Biology, University of Leicester, UK 2 Department of Cardiovascular Sciences, University of Leicester, UK 3 Joint first authors. INTRODUCTION Apis mellifera Bombus terrestris Biological function Chymotrypsin Chymotrypsin-1-like (LOC100648122) Upregulated in bumblebee non-reproductive workers (Pereboom et al., 2005) Gemini Upstream binding protein 1-like (LOC100650338) Upregulated in honeybee reproductive workers (Jarosch et al., 2011) Cabut Zinc finger protein 691-like (LOC100642767) Upregulated in honeybee non- reproductive workers (Cardoen et al., 2011) Ecdysone 20 monooxygenase Ecdysone 20 monooxygenase-like (LOC100649449) Upregulated in honeybee reproductive workers (Cardoen et al., 2011) Yolkless Vitellogenin receptor-like (LOC100649042) Upregulated in honeybee reproductive workers (Cardoen et al., 2011) Epidermal growth factor receptor Epidermal growth factor receptor like (LOC100645521) Upregulation of EGFR initiates ovary activation in queenless honeybee workers (Formesyn et al., 2014) Ribosomal Protein L26 Ribosomal Protein L26 like (LOC100648461) Differentially expressed in honeybee reproductive and non-reproductive workers (Thompson et al., 2007) Odorant receptor2 Or2 odorant receptor 2/Queen mandibular pheromone (QMP) co-receptor (LOC100631089) Upregulated in honeybee sterile workers (Grozinger et al., 2007) Dop3 D2-like dopamine receptor D2 like dopamine receptor (LOC100644210) Upregulated in honeybee non-reproductive workers (Vergoz, Lim & Oldroyd, 2012) Megator Megator TPR like nucleoprotein (LOC100645723) Upregulated in honeybee reproductive workers (Cardoen et al., 2011) Ecdysteroid regulated gene E93/Mblk-1 transcription factor Mushroom body large-type Kenyon cell specific protein 1-like (LOC100645656) Upregulated in honeybee reproductive workers (Cardoen et al., 2011) Ecdysone inducible gene L2/ImpL2 Neural/ectodermal development factor IMP-L2-like (LOC100645498) Upregulated in honeybee non-reproductive workers (Cardoen et al., 2011) Notes. NCBI gene IDs are in parentheses. chose twelve genes previously associated with worker reproduction in bees (see Table 1). We looked for polymorphisms in their exonic DNA in queens and their daughter workers using single strand conformation polymorphism (SSCP) and Sanger sequencing. SSCP relies on the principle that the electrophoretic mobility of a single-stranded DNA molecule is dependent on its structure (nucleotide sequence) and size. In the absence of the complementary strand, DNA becomes unstable and reanneals to itself to form conformations; hairpins, pseudoknots and triple helices (Nielsen, Novoradovsky & Goldman, 1995). These conformations vary according to the primary sequence of the molecule, such that a single nucleotide difference in DNA could dramatically affect the strand’s mobility through a gel due to its unique 3D structure. If we found that the workers possessed an allele from the queen and another allele not present in the queen, this allele must be from the father. That is, we identified a matrigene (allele from the mother) and patrigene (allele from the father) at this locus. INTRODUCTION Epigenetics refers to heritable changes in gene expression that do not involve DNA sequence alterations. Several recent reviews have heralded hymenopteran insects (ants, bees and wasps) as important emerging models for epigenetics (Glastad et al., 2011; Weiner & Toth, 2012; Welch & Lister, 2014; Yan et al., 2014). This is based mainly on data showing a fundamental role for methylation in their biology (Chittka, Wurm & Chittka, 2012). Methylation, the addition of a methyl group to a cytosine, is an epigenetic marker in many species (Glastad et al., 2011). Submitted 8 March 2015 Accepted 14 June 2015 Published 14 July 2015 Corresponding author Eamonn B. Mallon, ebm3@le.ac.uk Academic editor Leon Higley Additional Information and Declarations can be found on page 10 DOI 10.7717/peerj.1079 Copyright 2015 Amarasinghe et al. Distributed under Creative Commons CC-BY 4.0 OPEN ACCESS Submitted 8 March 2015 Accepted 14 June 2015 Published 14 July 2015 Corresponding author Eamonn B. Mallon, ebm3@le.ac.uk Academic editor Leon Higley Additional Information and Declarations can be found on page 10 DOI 10.7717/peerj.1079 Copyright 2015 Amarasinghe et al. Distributed under Creative Commons CC-BY 4.0 The recently sequenced genome of the bumblebee, Bombus terrestris displays a full complement of genes involved in the methylation system (Sadd et al., 2015). In a recent paper (Amarasinghe, Clayton & Mallon, 2014), we showed that methylation is important in worker reproduction in this bumblebee. We found methylation differences between the genomes of queenless reproductive workers and queenless non-reproductive workers. In a follow up experiment, queenless workers whose genomes had experimentally altered methylation (fed 5-aza-2′-deoxycytidine) were more aggressive and more likely to develop ovaries compared with control queenless workers. Previous work has found methylation associated with allele specific expression in a number of loci in the ants Camponotus floridanus and Harpegnathos saltator (Bonasio et al., 2012). Based on our result showing the importance of methylation in bumblebee worker reproduction, we searched for allele specific expression in worker reproduction genes. We Distributed under Creative Commons CC-BY 4.0 Table 1 Candidate genes selected from the literature search. Amarasinghe et al. (2015), PeerJ, DOI 10.7717/peerj.1079 Samples The queen and 5 randomly selected workers from each colony were used for SSCP analysis. Most candidate genes were tested in four different bumblebee colonies. Chymotrypsin, Gemini, Cabut and Yolkless were tested in colonies 1–4. Another four colonies (5–8) were used to test Epidermal growth factor receptor, Ribosomal protein L26, Odorant receptor 2, Dop3, Megator, Ecdysteroid regulated gene E93 (Mblk1) and Ecdysone inducible gene L2 (IMP-L2). Ecdysone 20 monooxygenase-like was tested in eight colonies. All qPCR data are based on bees from colony 5. DNA and RNA extraction and cDNA synthesis Bees were frozen in liquid nitrogen and then stored at −80 ◦C. Genomic DNA for SSCP analysis was extracted from each queen and respective worker bees using the Qiagen DNA Micro kit according to manufacturer’s instructions. Concentration of total genomic DNA was measured using the NanoDrop 1000 Spectrophotometer. A 30 mg sample of frozen tissue was ground with mortar and pestle on dry ice. RNA was extracted with the QIAGEN RNeasy Mini Kit according to manufacturer’s instructions. Any DNA contamination present in the above RNA extractions was removed according to Amplification Grade DNase I Kit protocol (Sigma-Aldrich), prior to the synthesis of cDNA. Concentrations of DNase treated RNA was determined by the NanoDrop 1000 spectrophotometer. cDNA was synthesized from a 8 µl sample of RNA using the Tetro cDNA synthesis Kit (Bioline) as per manufacturer’s instructions. Synthesized cDNA was stored at −80 ◦C. Identification of candidate genes and designing primers Identification of candidate genes and designing primers Twelve social insect genes previously associated with differential expression in queens, reproducing workers and non-reproducing workers were selected via a literature search (Table 1). Sequences for all selected candidate genes were obtained from Apis mellifera genome data, available in NCBI. Apis mellifera data was BLASTed against the Bombus terrestris Nucleotide library (NCBI) in order to find the homolog in Bombus terrestris. Primers were designed to the exonic regions using Geneious Pro (version 5.5.6) and primer 3 version 0.4.0 (http://frodo.wi.mit.edu). The focus was on exonic regions to ensure that the same loci was present in the cDNA for the allele specific qPCR analysis. Amarasinghe et al. (2015), PeerJ, DOI 10.7717/peerj.1079 INTRODUCTION If we found this, we carried out an allele specific qPCR to ascertain if this locus displayed allele specific expression. Amarasinghe et al. (2015), PeerJ, DOI 10.7717/peerj.1079 2/13 Allele specific PCR Allele specific PCR was used to confirm the maternal and paternal alleles identified during heterozygote analysis. Allele specific primers were designed using Batch primer 3 program (http://probes.pw.usda.gov/batchprimer3/) to cover the heterozygotic nucleotide positions identified above. Two forward primers specific to either maternal (F1) or paternal (F2) allele sequences and a common reverse primer were designed. Genomic DNA of the queen and 5 heterozygous workers in each colony, were PCR amplified with these allele specific primers (Table 2). PCR products were checked on a 3% agarose gel. When using allele specific primers, only the allele which includes the relevant snp would be amplified. Primers which amplified the snp region successfully were used for qPCR analysis. SSCP analysis SSCP analysis was carried out according to Gasser et al. (2007) using GMA wide mini S-2x25 gels (Elchrom Scientific). Sample denaturing solution was prepared by mixing 990 µl of 95% formamide with 10 µl of 1 M NaOH just prior to use. 4 µl of the PCR product was denatured with 7 µl of denaturing mixture, incubated in a thermocycler at 94 ◦C for 10 min and immediately chilled on ice for 5 min. The temperature of the running buffer (1x TAE) in the Origins gel tank (Elchrom Scientific) was kept constant at 9 ◦C. 7 µl of the denatured PCR product was mixed with 2 µl of Elchrom loading dye and loaded in to a well on the gel. The gels were run at 72 V. The electrophoretic running times were varied depending on the fragment size; 10 h for 150–200bp fragment length, 12 h for 200–250bp fragment length, 15 h for 250–350bp fragment length and 17 h for 350–450bp fragment length. Following electrophoresis, the gels were stained for 30 min with SybrGold (Invitrogen) (1:10,000 diluted in TAE) and destained with 100 ml of 1x TAE buffer for a further 30 min. If a polymorphic banding pattern among the queen and her 5 workers was observed during SSCP, another SSCP was run to confirm the reproducibility of those results. The genomic DNA of those queen and worker bees were amplified with their respective If a polymorphic banding pattern among the queen and her 5 workers was observed during SSCP, another SSCP was run to confirm the reproducibility of those results. during SSCP, another SSCP was run to confirm the reproducibility of those results. The genomic DNA of those queen and worker bees were amplified with their respective primers (see Table S1) and PCR products were sent for commercial clean up and Sanger sequencing. All sequencing results were blasted against NCBI, Bombus terrestris nucleotide library to verify if the correct sequence was amplified. Sequencing results were analyzed using the heterozygote analysis module in Geneious version 7.3.0 to identify heterozygotic nucleotide positions. PCR amplifications For each primer set, a 25 µl reaction volume (60 ng of DNA, 12.5 µl YB-Taq 2x Buffer, 1.5 µl of each forward and reverse primer (10 µlM/µl), 1 µl of 10 mM MgCl2 and 6.5 µl of dH20) was run using the following conditions: an initial denaturation for 5 min at 94 ◦C, 30 cycles of 30 s at 94 ◦C, 30 s each at the relevant annealing temperature followed by a final exten- sion of 10 min at the relevant extension temperature and a holding step of 4 ◦C. The se- quences and annealing and extension temperatures used for each primer set are in Table S1. Prior to SSCP analysis, each PCR product (10 µl) was checked on a 3% agarose gel. If the correct size of amplicon was obtained, then the rest of the sample (15 µl) was used for SSCP. Amarasinghe et al. (2015), PeerJ, DOI 10.7717/peerj.1079 3/13 Amarasinghe et al. (2015), PeerJ, DOI 10.7717/peerj.1079 Allele specific quantitative PCR Reference primers were designed according to Gineikiene, Stoskus & Griskevicius (2009). A common forward primer was designed to the same target heterozygote sequence, upstream of the heterozygote nucleotide position, leaving the same common reverse primer previ- ously used with allele specific primers (see reference sequences in Table 2). The reference primers measure the total expression of the gene, whereas the allele specific primers mea- sure the amount of expression due to the allele. Thus the ratio between the allele specific expression and reference locus expression would be the relative expression due to the allele. Amarasinghe et al. (2015), PeerJ, DOI 10.7717/peerj.1079 4/13 4/13 Table 2 Allele specific primers used for gene expression analysis. Gene Heterozygote position Primer sequence (5′-3′) TA (◦C) Product size (bp) F1: GCGGAAGCCGTCAGG F2: TTAGCGGAAGCCGTCAGA Ecdysone 20 monooxygenase like 48 (A/G) R: GCGAGGCCGTAAAGTGTAT 58 34 F: GATTTAGCGGAAGCCGTCAG Ecdysone 20 monooxygenase like internal reference R: GCGAGGCCGTAAAGTGTAT 59 36 F1: ACTTGCCAAGCCAAGTCTG F2: CACTTGCCAAGCCAAGTCTA IMP-L2-like 253 (A/G) R: TTCGAGCCACTTCCTTTTCG 59.5 205 F: CTACACTTGCCAAGCCAAGTCT IMP-L2-like internal reference R: TTCGAGCCACTTCCTTTTCG 59.5 207 Notes. The snp present is located at the ′ end of each forward primer (marked in red). F1, Forward primer 1; F2, Forward primer 2; R, Common reverse primer; TA, Annealing temperature. Notes. The snp present is located at the ′ end of each forward primer (marked in red). F1, Forward primer 1; F2, Forward primer 2; R, Common reverse primer; TA, Annealing temperature. Each heterozygous locus was run for 3 different reactions; maternal (F1), paternal (F2) and reference (Table 2). Three replicate samples were run for each reaction. All reactions were prepared by the Corbett robotics machine, in 96 well qPCR plates (Thermo Scientific, UK). The qPCR reaction mix (20 µl) was composed of 1 µl of diluted cDNA (50 ng/µl), 1 µl of forward and reverse primer (5 µM/µl each, Table 2), 10 µl 2x SYBR Green JumpStart Taq ReadyMix (Sigma Aldrich, UK) and 7 µl ddH20. Samples were run in a PTC-200 MJ thermocycler. The qPCR profile was; 4 min at 95 ◦C denaturation followed by 40 cycles of 30 s at 95 ◦C, 30 s at the relevant annealing temperature (Table 2) and 30 s at 72 ◦C and a final extension of 5 min at 72 ◦C. Forward primers are different, both in their terminal base (to match the snp) and in their length. Allele specific quantitative PCR It is entirely possible that they may amplify more or less efficiently even if there was no difference in amount of template (Pfaffl, 2001). To test for this we repeated all qPCRs with genomic DNA 1 µl of diluted DNA (20 ng/ µl) from the same bees as the template. We would expect equal amounts of each allele in the genomic DNA. We also measured efficiency of each reaction as per Liu & Saint (2002). Amarasinghe et al. (2015), PeerJ, DOI 10.7717/peerj.1079 SSCP analysis Exon coverage for each gene is given in Table 3. We found no polymorphisms in nine genes out of the twelve candidate genes tested (see Table 3). Figure 1 shows representative examples of these gels with the queen and her workers sharing the same banding pattern. Exon coverage for each gene is given in Table 3. We found no polymorphisms in nine genes out of the twelve candidate genes tested (see Table 3). Figure 1 shows representative examples of these gels with the queen and her workers sharing the same banding pattern. Compared with the queen, workers in colony 3, 5 and 7 showed a heterozygous banding pattern in 3 genes; ecdysone 20-monooxygenase-like (ecdysone 20-monooxygenase-like), IMPL-2-like and MBLK1-like (Fig. 2). g g g Compared with the queen, workers in colony 3, 5 and 7 showed a heterozygous banding pattern in 3 genes; ecdysone 20-monooxygenase-like (ecdysone 20-monooxygenase-like), IMPL-2-like and MBLK1-like (Fig. 2). Amarasinghe et al. (2015), PeerJ, DOI 10.7717/peerj.1079 Data analysis Median Ct was calculated for each set of three technical replicates. A measure of relative expression (ratio) was calculated for each parental allele in each worker bee as follows: ratiomaternal = E−Ctmaternal maternal E−Ctreference reference (1) ratiopaternal = E −Ctpaternal paternal E−Ctreference reference . (2) ratiomaternal = E−Ctmaternal maternal E−Ctreference reference ratiopaternal = E −Ctpaternal paternal E−Ctreference reference . (1) (2) Amarasinghe et al. (2015), PeerJ, DOI 10.7717/peerj.1079 5/13 Figure 1 The queen (Q) and 5 workers (W1–W5) are represented in each colony. SSCP gel results of six genes (A–F) with no queen-worker variations (homozygous banding patterns). Figure 1 The queen (Q) and 5 workers (W1–W5) are represented in each colony. SSCP gel results of six genes (A–F) with no queen-worker variations (homozygous banding patterns). Figure 1 The queen (Q) and 5 workers (W1–W5) are represented in each colony. SSCP gel results of six genes (A–F) with no queen-worker variations (homozygous banding patterns). E is the median efficiency of each primer set (Liu & Saint, 2002; Pfaffl, 2001). Matched paired t-tests was performed to check if the allele specific expression values are significantly different among the two parental alleles. All statistical analysis was carried out using R (3.1.0) (R Core Team , 2015). Sequences of polymorphic loci Gene name Coverage (%) Polymorphism Chymotrypsin-1-like 92 Absent Upstream-binding protein 1-like 93 Absent Zinc finger protein 691-like 70 Absent Vitellogenin receptor-like 30 Absent Epidermal growth factor receptor like 21 Absent 60S Ribosomal Protein L26 like 32 Absent Or2 odorant receptor 2 25 Absent D2 like dopamine receptor 54 Absent Megator TPR like nucleoprotein 17 Absent Ecdysone 20-monooxygenase-like 37 Present Mushroom body large-type Kenyon cell-specific protein 1-like 35 Present Neural/ectodermal development factor IMP-L2-like 47 Present Gene name Coverage (%) Polymorphism Chymotrypsin-1-like 92 Absent Upstream-binding protein 1-like 93 Absent Zinc finger protein 691-like 70 Absent Vitellogenin receptor-like 30 Absent Epidermal growth factor receptor like 21 Absent 60S Ribosomal Protein L26 like 32 Absent Or2 odorant receptor 2 25 Absent D2 like dopamine receptor 54 Absent Megator TPR like nucleoprotein 17 Absent Ecdysone 20-monooxygenase-like 37 Present Mushroom body large-type Kenyon cell-specific protein 1-like 35 Present Neural/ectodermal development factor IMP-L2-like 47 Present Figure 2 SSCP allelic polymorphism in IMP-L2-like, MBLK1-like and Ecdysone 20-monooxygenase- like. Figure 2 SSCP allelic polymorphism in IMP-L2-like, MBLK1-like and Ecdysone 20-monooxygenase- like. Figure 2 SSCP allelic polymorphism in IMP-L2-like, MBLK1-like and Ecdysone 20-monooxygenase- like. Allele specific PCR Allele specific primers designed for ecdysone 20-monooxygenase-like and IMP-L2-like worked successfully with genomic DNA and cDNA to produce the expected fragment lengths. They were used for gene expression analysis in the next section. Amplification of MBLK1-like using allele specific primer sets was unsuccessful possibly due to the large number of snps. Thus, we did not continue with qPCR analysis for MBLK1-like. Amarasinghe et al. (2015), PeerJ, DOI 10.7717/peerj.1079 Sequences of polymorphic loci We sequenced the three loci showing heterozygous banding patterns in SSCP gels. In ecdysone 20-monooxygenase-like, the queen sequence is homozygous (Fig. 2). At the snp (2,474th base pair of LOC100649449) the queen has a guanine (G), while all of her workers show double peaks corresponding to both guanine (G) and adenine (A) bases (see Table S2 for sequences). From this we identified the matrigene as containing guanine and the patrigene as containing adenine. A similar SSCP banding pattern was found for IMP-L2-like (Fig. 2). Again the queen had a guanine whereas workers contained a guanine and an adenine, this time at the 5,130th base pair of LOC100645498 (Table S2). MBLK1-like has several snps in the amplified region (Fig. 2 and Table S2). Amarasinghe et al. (2015), PeerJ, DOI 10.7717/peerj.1079 Table 3 SSCP exon coverage. Percentage exon coverage for each gene was calculated as the sum of all tested amplicon lengths as a fraction of the total length of mRNA per gene. Gene name Coverage (%) Polymorphism Chymotrypsin-1-like 92 Absent Upstream-binding protein 1-like 93 Absent Zinc finger protein 691-like 70 Absent Vitellogenin receptor-like 30 Absent Epidermal growth factor receptor like 21 Absent 60S Ribosomal Protein L26 like 32 Absent Or2 odorant receptor 2 25 Absent D2 like dopamine receptor 54 Absent Megator TPR like nucleoprotein 17 Absent Ecdysone 20-monooxygenase-like 37 Present Mushroom body large-type Kenyon cell-specific protein 1-like 35 Present Neural/ectodermal development factor IMP-L2-like 47 Present Figure 2 SSCP allelic polymorphism in IMP-L2-like, MBLK1-like and Ecdysone 20-monooxygenase- like. Table 3 SSCP exon coverage. Percentage exon coverage for each gene was calculated as the sum of all tested amplicon lengths as a fraction of the total length of mRNA per gene. Table 3 SSCP exon coverage. Percentage exon coverage for each gene was calculated as the sum of all tested amplicon lengths as a fraction of the total length of mRNA per gene. Allele specific qPCR The allele specific primer sets for ecdysone 20-monooxygenase-like showed no difference in their ability to amplify genomic DNA (paired t-test: t = 0.4815, df = 4, p-value = 0.6553, Maternal primers Ct = 36.73 ± 2.494, Paternal primers Ct = 36.27 ± 1.792 (mean ± standard deviation)). For IMP-L2-like, the two primer sets did show a significant difference in efficiency to amplify genomic DNA (paired t-test: t = 7.062, df = 4, p-value = 0.002121, Maternal primers Ct = 35.83 ± 1.463, Paternal primers Ct = 32.49 ± 1.327 Amarasinghe et al. (2015), PeerJ, DOI 10.7717/peerj.1079 7/13 Figure 3 Expression (measured as ratio of allelic to reference primer expression (see ‘Methods’)) differences between maternal and paternal alleles of ecdysone 20-monooxygenase-like. The first plot shows the expression data as individual dots. The diagonal lines join data from the same bee. Boxplots represent the distributions. The second plot shows the proportion of maternal to paternal expression as individual dots. Figure 3 Expression (measured as ratio of allelic to reference primer expression (see ‘Methods’)) differences between maternal and paternal alleles of ecdysone 20-monooxygenase-like. The first plot shows the expression data as individual dots. The diagonal lines join data from the same bee. Boxplots represent the distributions. The second plot shows the proportion of maternal to paternal expression as individual dots. (mean ± standard deviation)). As Ct decreases with increasing copy number, the paternal primers amplified better than the maternal set. To control for this difference in primer efficiency we used a modification of the pfaffl method to calculate a measure of expression (Liu & Saint, 2002; Pfaffl, 2001). This includes efficiency in its calculations (see ‘Methods’). The patrigene showed significantly increased expression compared to the matrigene in ecdysone 20-monooxygenase-like (Fig. 3) (paired t-test: t = −7.517, df = 4, p-value = 0.001676). For IMP-L2-like, the matrigene was more expressed than the patrigene (Fig. 4) (paired t-test: t = 3.409, df = 4, p-value = 0.02705). Amarasinghe et al. (2015), PeerJ, DOI 10.7717/peerj.1079 DISCUSSION Using a candidate gene approach we found evidence for allele specific expression in the bumblebee, Bombus terrestris. Out of twelve genes examined during this study, we found exonic variation in only three genes; MBLK1-like, IMP-L2-like and ecdysone 20-monooxygenase-like. Of these we were able to carry out allele specific qPCR on IMP-L2-like and ecdysone 20-monooxygenase-like. We found allele specific expression in ecdysone 20-monooxygenase-like and IMP-L2-like. Use of SSCP to find exonic variation is challenging. SSCP detects variation in fragments up to 500bp size with a high resolution of 1bp. However, the sensitivity of SSCP decreases when the fragment length exceeds 200bp (Weber et al., 2005). Thus medium length fragments around 200bp were used for this analysis. Covering the full exome using SSCP would be a time consuming and labour intensive process. Added to this, variation in protein coding exons is expected to be rarer than in introns (Castle, 2011). One possibility would be to look at the exons in untranslated regions (UTRs), which would be expected to be more variable than protein coding exons (Araujo et al., 2012; Lytle, Yario & Steitz, 2007). Amarasinghe et al. (2015), PeerJ, DOI 10.7717/peerj.1079 8/13 Figure 4 Expression (measured as ratio of allelic to reference primer expression (see ‘Methods’)) differences between maternal and paternal alleles of Ecdysone-inducible gene L2 (IMP-L2-like). The first plot shows the expression data as individual dots. The diagonal lines join data from the same bee. Boxplots represent the distributions. The second plot shows the proportion of maternal to paternal expression as individual dots. Figure 4 Expression (measured as ratio of allelic to reference primer expression (see ‘Methods’)) differences between maternal and paternal alleles of Ecdysone-inducible gene L2 (IMP-L2-like). The first plot shows the expression data as individual dots. The diagonal lines join data from the same bee. Boxplots represent the distributions. The second plot shows the proportion of maternal to paternal expression as individual dots. Figure 4 Expression (measured as ratio of allelic to reference primer expression (see ‘Methods’)) differences between maternal and paternal alleles of Ecdysone-inducible gene L2 (IMP-L2-like). The first plot shows the expression data as individual dots. The diagonal lines join data from the same bee. Boxplots represent the distributions. The second plot shows the proportion of maternal to paternal expression as individual dots. Our expression analysis used the bees’ whole bodies. Amarasinghe et al. (2015), PeerJ, DOI 10.7717/peerj.1079 DISCUSSION Therefore gene expression patterns observed during this analysis should represent the overall expression of all body tissues. However, potentially it means allele specific expression which is only found in some tissues would be masked by the overall response. Our expression analysis used the bees’ whole bodies. Therefore gene expression patterns observed during this analysis should represent the overall expression of all body tissues. However, potentially it means allele specific expression which is only found in some tissues would be masked by the overall response. We found allele specific expression in Ecdysone 20 monooxygenase. Ecdysone 20 monooxygenase catalyses the reaction which turns ecdysteroid ecdysone into 20- hydroxyecdysone, also an ecdysteroid. An up-regulation of ecdysone 20-monooxygenase- like was observed in egg laying honeybee workers compared to non-reproductive work- ers (Cardoen et al., 2011). Generally, ecdysteroids have been identified as key regulators of B. terrestris worker reproduction (Geva, Hartfelder & Bloch, 2005). Ecdysteriods are key compounds involved in ovary activation, regulating agonistic behaviour and establishing the dominance hierarchy in workers and queens (Geva, Hartfelder & Bloch, 2005). We also found allele specific expression in IMP-L2-like. In honeybees, this gene is linked with reproductive inhibition of workers. It functions similarly to an insulin like peptide and negatively regulates insulin signaling pathways to repress ovary activation (Cardoen et al., 2011; Grozinger et al., 2007). Our analysis found allele specific expression in two worker reproduction genes. We are interested in this as an example of epigenetics. However, allele specific expression is known to be caused by a number of genetic (i.e., cis-acting inherited variation) as well as epigenetic (e.g., genomic imprinting) processes (Palacios et al., 2009). As our results are based on a single genetic line (colony) we are unable to say whether the examples of allele specific expression we found are due to epigenetic or genetic causes. Given this, it is still interesting to note that the expression patterns of both ecdysone 20-monooxygenase-like and IMP-L2-like are consistent with those predicted for genomic Amarasinghe et al. (2015), PeerJ, DOI 10.7717/peerj.1079 imprinted genes involved in worker reproduction in a singly mated social insect colony (Queller, 2003). Queller (2003) used Haig’s kinship theory for the evolution of genomic imprinting (Haig, 2000) to predict the imprinting patterns of genes involved in various functions under various social contexts in the social insects. Competing Interests The authors declare there are no competing interests. Amarasinghe et al. (2015), PeerJ, DOI 10.7717/peerj.1079 Grant Disclosures The following grant information was disclosed by the authors: NERC: NE/H010408/1. Funding This work was financially supported by NERC grant no. NE/H010408/1 to EBM. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ACKNOWLEDGEMENT Thanks to Sally Adams for discussions. DISCUSSION He predicted that genes that are associated with the initiation of worker reproduction (e.g., ecdysone 20-monooxygenase-like) should be paternally expressed in social insect species such as B. terrestris with singly-mated, monogynous (one queen), queenright (queen still alive) colonies. Ecdysone 20-monooxygenase-like’s expression is consistent with increased pa- ternal expression. Reciprocally we would expect a gene that inhibits worker reproduction (e.g., IMP-L2-like) to be maternally expressed. IMP-L2-like’s expression is consistent with increased maternal expression. Fascinating as this is, it must be tempered with the proviso that, as previously stated, this work was carried out on a single genetic line so cannot differentiate epigenetic from genetic causes. Clearly the candidate gene approach is limited in its application. Next generation sequencing technology allows gene expression analysis at genome-wide scale (RNA-seq). Several recent papers have applied RNA-seq to search, without bias, for novel imprinted genes in mammals (Okae et al., 2012; DeVeale, Van der Kooy & Babak, 2012; Gregg et al., 2010; Wang & Clark, 2014) and flowering plants (Gehring, 2013). Our results suggest that this could be a potentially fruitful avenue for research in social insects. REFERENCES Amarasinghe HE, Clayton CI, Mallon EB. 2014. 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https://openalex.org/W2998515836	https://journal.uinjkt.ac.id/index.php/arabiyat/article/download/12310/pdf	English	null	PERFORMANCE OF ARABIC EDUCATION LECTURERS IN DEVOTION OF LANGUAGE LEARNING	Arabiyat : Jurnal Kebahasaaraban dan Pendidikan Bahasa Arab/Arabiyat: Jurnal Pendidikan Bahasa Arab dan Kebahasaaraban	2,019	cc-by-sa	7,698	Arabiyat : Jurnal Pendidikan Bahasa Arab dan Kebahasaaraban Vol. 6 No. 2, December 2019, 272-287 P-ISSN: 2356-153X; E-ISSN: 2442-9473 doi: http://dx.doi.org/10.15408/a.v6i2.12310 Arabiyat : Jurnal Pendidikan Bahasa Arab dan Kebahasaaraban Vol. 6 No. 2, December 2019, 272-287 P-ISSN: 2356-153X; E-ISSN: 2442-9473 doi: http://dx.doi.org/10.15408/a.v6i2.12310 Muhammad Qodri Universitas Islam Negeri Sulthan Thaha Saifuddin Jambi, Indonesia Jl. Arif Rahman Hakim No. 111, Kec. Telanaipura, Kota Jambi, 36361, Indonesia Corresponding E-mail: muhammadqodri@uinjambi.ac.id Muhammad Qodri Universitas Islam Negeri Sulthan Thaha Saifuddin Jambi, Indonesia Jl. Arif Rahman Hakim No. 111, Kec. Telanaipura, Kota Jambi, 36361, Indonesia Corresponding E-mail: muhammadqodri@uinjambi.ac.id Abstract T This article aims to find out how the performance of lecturers in designing, implementing and evaluating learning language proficiency courses. This article uses a descriptive qualitative approach. data collection through interviews, observation, and documentation. Data collected using snowball sampling techniques and data collected were analyzed through the steps of data reduction, data presentation, and drawing conclusions. The findings of this article are: performance of Arabic education lecturers faculty of Tarbiyah in devotion of language learning that lecturer has not maximized the potential in giving lectures, coupled with the preparation of SAP courses that are lacking this is seen in: 1) the design of learning made by lecturers listening and reading in the form of SAP, only briefly explains about several components, namely the identity of the course, learning outcome indicators, lecture method, assessment, and bibliography, mentions several reading sources used in the listening learning process and reading; 2) the implementation of learning include: preliminary activities, core activities, and closing activities; 3) the evaluation of learning outcomes is an activity of measuring the level of success and ability of students in the learning process. PERFORMANCE OF ARABIC EDUCATION LECTURERS IN DEVOTION OF LANGUAGE LEARNING Muhammad Qodri Universitas Islam Negeri Sulthan Thaha Saifuddin Jambi, Indonesia Jl. Arif Rahman Hakim No. 111, Kec. Telanaipura, Kota Jambi, 36361, Indonesia Corresponding E-mail: muhammadqodri@uinjambi.ac.id Arabic lecturer, lecturer performance, Arabic education, language learning devotion Arabic lecturer, lecturer performance, Arabic education, language learning devotion ensure the achievement of instructional and institutional goals, and 5) achieve learning effectiveness. The performance of lecturers in learning includes three major components, namely the skills in designing, implementing and evaluating learning. At least there are several reasons underlying the discussion of the performance of lecturers in this learning. According to Davis, the task of teaching is a professional activity that requires high-level skills and includes making decisions intended to facilitate the teaching and learning process, to improve the ability and acquisition of student learning outcomes based on predetermined goals. Furthermore, according to Davis, lecturers who function as learning designers are required to be able to realize their performance well in this learning process.1 From the description above, it can be seen that every lecturer needs to show the right decisions, such as what to do, who does it, how to do it, and more importantly why it needs to be done. The accuracy in making decisions on these problems will have a positive impact on the performance of lecturers in learning. To be able to make the right decisions, lecturers need to have an adequate knowledge base on the characteristics of students, theories and principles of learning, design, and development of learning systems, selection of effective learning methods, assessment of student learning outcomes, problems that may be faced, and strategies in mitigating it.2 The observations of researchers as PBA lecturers at the Tarbiyah and Teaching Faculty of UIN STS Jambi, language proficiency courses (listening and reading) which have been fostered by several lecturers have been running on their own so that they realize their performance themselves. self with the characteristics of each performance. The design, implementation, and evaluation of these courses vary from one lecturer to another lecturer, even though they hold the same subject. This uniqueness is the reason for the need to be investigated, to provide a description of each performance in learning this language proficiency course, as well as expressing the underlying arguments decided and carried out by each lecturer. Determination of tarbiyah and teacher faculties, as well as language proficiency courses as the object of this research, besides because the researchers themselves are lecturers in the faculty, also because there has not been a significant change in learning that has an impact on improving quality in the learning process. 1 Hamzah B. Uno, Profesi Kependidikan, (Jakarta: Bumi Aksara, 2008), 77-80. 2 Wor. K. Davis, The Management of Learning, (London: McGraw Hill Book Company, 1971), 114. Introduction The performance of lecturers in the implementation of learning can be determined at a level through their competence in carrying out the tasks in the performance. Lecturer performance in this learning has a large influence and can determine the level of quality of graduates. The lecturers' pedagogical competencies in learning include the ability and skills to design learning, develop and implement learning strategies, evaluate and perfect learning. The learning design basically functions to 1) strengthen the learning process, 2) know immediately the success rate of the learning process, 3) increase the confidence and enthusiasm of students, 4) Arabiyât Jurnal Pendidikan Bahasa Arab dan Kebahasaaraban, 6(2), 2019 ensure the achievement of instructional and institutional goals, and 5) achieve learning effectiveness. ensure the achievement of instructional and institutional goals, and 5) achieve learning effectiveness. , f p , (J , ), Wor. K. Davis, The Management of Learning, (London: McGraw Hill Book Company, 1971), 114. Hamzah B. Uno, Profesi Kependidikan, (Jakarta: Bumi Aksara, 2008), 77-80. Wor K Davis The Management of Learning (London: McGraw Hill Book Company 1971) 114 3 Noah Webster, Websters's New Twentieth Century Dictionary of English Language, (London: William Collins Publisher, 1980), 156. 4 Robert S.Zais, Curriculum Principles, and Foundation, (Newyork: Harper & Row Publisher, 1976), 84. 5 Yusrizal, “Performance Assessment of State Senior High School Teachers Aged 56 Years and Above”, International Journal of Instruction, Vol.11, No.1, 2018, 38. 6 Syafruddin Nurdin, Guru Profesional dan Implementasi Kurikulum, (Jakarta: Ciputat Pers, 2002), 42. 7 Arif Partono Prasetio, “Lecturers’ Professional Competency and Students’ Academic Performance in Indonesia Higher Education”, International Journal of Human Resource Studies, Vol. 7, No. 1, 2017, 88. 8 Suryaman, “Indonesian Private University Lecturer Performance Improvement Model to Improve a Sustainable Organization Performance”, International Journal of Higher Education, Vol. 7, No. 1, 2018, 61. Arabiyât Jurnal Pendidikan Bahasa Arab dan Kebahasaaraban, 6(2), 2019 Arabiyât Jurnal Pendidikan Bahasa Arab dan Kebahasaaraban, 6(2), 2019 8 Suryaman, “Indonesian Private University Lecturer Performance Improvement Model to Improve a Sustainable Organization Performance”, International Journal of Higher Education, Vol. 7, No. 1, 2018, 61. Theoretical Framework Lecturer Performance in Learning Lecturer Performance in Learning ensure the achievement of instructional and institutional goals, and 5) achieve learning effectiveness. Therefore, as a first step, it is necessary to have a scientific description of the performance of lecturers in learning this language proficiency course, so that the lecturers can be motivated to further improve the quality of their performance in the whole series of learning. ARABIYAT, ISSN: 2356-153X, E-ISSN: 2442-9473 273 Arabiyât Jurnal Pendidikan Bahasa Arab dan Kebahasaaraban, 6(2), 2019 Arabiyât Jurnal Pendidikan Bahasa Arab dan Kebahasaaraban, 6(2), 2019 lecturer as a lecturer in a university is all the behavior of the lecturer who deals with the implementation of his functions and duties in accordance with the criteria determined by the university concerned. The behavior of the lecturer as teaching staff in higher education in implementing learning design is very influential and determines the events of implementation and evaluation of learning outcomes. lecturer as a lecturer in a university is all the behavior of the lecturer who deals with the implementation of his functions and duties in accordance with the criteria determined by the university concerned. The behavior of the lecturer as teaching staff in higher education in implementing learning design is very influential and determines the events of implementation and evaluation of learning outcomes. 9 Morrison GR & S.M. Ross, Designing Effective Instruction, (USA: Macmillan College Publishing Company, Inc. 1994), 76. Learning Design g g The main activities of system development and learning design in order to design the learning include: a. Stage identify 1. Identify learning needs and write general learning objectives 2. Conduct a learning analysis 3. Identify early human behavior and characteristics. b. Development phase 1. Formulate specific learning objectives 2. Develop the benchmark reference test items 3. Develop learning strategies 4. Develop learning materials or materials. p g c. The stage of evaluating and revising is stated by arranging the design and carrying out a formative evaluation which includes revision activities.9 c. The stage of evaluating and revising is stated by arranging the design and carrying out a formative evaluation which includes revision activities.9 Based on some of the descriptions above, it can be stated that what must be included in the design of learning is an effort to develop and prioritize learning objectives, both long-term goals and short-term goals, which include: 1) identifying certain prerequisite skills and knowledge needed to achieve purpose, 2) designing learning to encourage student mastery of prerequisite skills, 3) preparing written learning units to support achievement of learning objectives, 4) determining learning strategies to be used, 5) stating levels of cognitive learning, understanding, application, analysis, synthesis, and evaluation, 6) designing the right evaluation. If the series of performance is realized properly, it will produce a Design of Teaching and Learning Activities (RKBM) or learning design that should be prepared and will be carried out by the teaching staff, thus enabling an effective and efficient learning system. Lecturer Performance in Learning Performance according to the understanding expressed by experts is basically the same. The difference is only found in their different perspectives. This definition of performance is nothing but the translation is performed. According to Webster that "performance is the ability to perform; capacity to achieve the desired result".3 According to Robert that "performance is put out derived from processes, human or otherwise”.4 From these two definitions can be drawn an understanding that performance is the ability to do something to achieve a predetermined goal or a result of the implementation of a person's work process. Performance is a decisive mean in achieving the goals of an institution or a company; however, this is not easy to do, because many factors affect the high and the low performance.5 Syafruddin argues that performance is an ability to carry out tasks or work in accordance with the attitudes, knowledge and skills and motivation of employees.6 The characteristics of the performance in question are carrying out tasks in accordance with the expectations of an organization that they embrace, using available office equipment, having high enthusiasm, having good cooperative relations with superiors as well as with colleagues, can overcome problems related to routine tasks carried out every day. Performance is one's level of success in executing a job at a certain period.7 Performance is defined as the ability to work in terms of quality and quantity. This means that the performance is a feat achieved by a person working both in quality and quantity that are served by an employee in performing their duties in accordance with the responsibilities given to him. Relation to lecturer profession, the lecturer's performance can be interpreted as a result of the quality and quantity of work achieved a lecturer in implementing the Tri Dharma University that includes education and teaching, research, and devotion to the public in accordance with the responsibilities given to him.8 Based on the description above, it can be understood that performance is all the behavior of a person in carrying out the functions and duties of a job assigned to him. In connection with this understanding, what is meant by the performance of a ARABIYAT, ISSN: 2356-153X, E-ISSN: 2442-9473 274 10 Gráinne Conole & Karen Fill, “A learning design toolkit to create pedagogically effective learning activities, A learning design toolkit to create pedagogically effective learning activities”, Journal of Interactive Media in Education, Vol. 8, 2005, 7-8. 11 C.M Reigeluth dan F.S.Stein, The Elaboration Theory of Instruction, (Hillsdale, N.J : Lawrence Erlbaum Associates, t.t.), 125. 12 Degeng IN, Ilmu Pengajaran , Taksonomi, Variable, (Jakarta : P2LPTK, 1991), 83. 13 AL Hough, An Approach to Curriculum Evaluation, (Colombo Plan Staff College For Technician, 1976), 137. Arabiyât Jurnal Pendidikan Bahasa Arab dan Kebahasaaraban, 6(2), 2019 specify the type of task, the techniques used, associated tools and resources, the interaction and roles of those involved and the assessments associated with the learning activity.10 Each lecturer has his own way to determine his learning activities, with the intention of facilitating students to learn and improve the achievement of set goals. Each method was chosen on the basis of confidence in the success of the student and his success in teaching. The selection may be based on certain theories in learning. specify the type of task, the techniques used, associated tools and resources, the interaction and roles of those involved and the assessments associated with the learning activity.10 Each lecturer has his own way to determine his learning activities, with the intention of facilitating students to learn and improve the achievement of set goals. Each method was chosen on the basis of confidence in the success of the student and his success in teaching. The selection may be based on certain theories in learning. According to Reigeluth and Merrill that to develop a learning theory needed clarification that is more detailed and very adequate as the basis for the development of learning theory. There are three main variables that are interrelated learning for development, namely: 1) learning conditions, 2) learning methods, 3) learning outcomes.11 Of the three main variables of learning, according to Degeng, the most decisive quality of learning is the learning method variable, as well as this variable requires the performance of lecturers as teaching staff in higher education in taking policies to implement learning strategies in the implementation of learning.12 Another study of learning implementation strategies was put forward by Hough which stated that there were nine sequences of learning activities, namely: 1) giving motivation or attracting attention, 2) explaining learning objectives to students, 3) reminding competency prerequisites, 4) giving stimulus, 5) giving learning instructions, 6) giving rise to student appearance, 7) giving feedback, 8) assessing skills, 9) concluding.13 The above study explains that the learning implementation strategy relates to the learning process approach in managing learning activities systematically so that it can facilitate students to learn, and the content of the lessons can be mastered effectively and efficiently. Learning Implementation At the heart of the toolkit is the notion of a learning activity, which we define as consisting of three elements; a) The context within which the activity occurs, this includes the subject, level of difficulty, the intended learning outcomes and the environment within which the activity takes place, b) The learning and teaching approaches adopted, including the theories and models, and c) The tasks are undertaken, which ARABIYAT, ISSN: 2356-153X, E-ISSN: 2442-9473 275 14 Wor. K. Davis, The Management of Learning, (London : McGraw Hill Book Company, 1971), 93. 15 Donald L. Rubin and Paul H. Matthews, “Learning Outcomes Assessment: Extrapolating from Study Abroad to International Service-Learning”, Journal of Higher Education Outreach and Engagement, Vol. 17, No. 2, 2013, 67-80. 16 Soekartawi, Meningkatkan Rancangan Instruksional Untuk Memperbaiki Kualitas Belajar Mengajar, (Jakarta: Raja Grafindo Persada, 1995), 86. b. Functions and Objectives of Learning Outcomes According to Sudjana the results of learning are carried out to 1) describe student learning skills so that they can know the strengths and weaknesses in the various fields of study, 2) know the success of the teaching and learning process, how far it is in changing students' behavior towards learning objectives expected, 3) determine the follow-up of the assessment results, and 4) provide accountability from the institution to interested parties. a. Concept of Evaluating Learning Outcomes a. Concept of Evaluating Learning Outcomes The evaluation concept described by Seels and Richey is the process of determining the suitability of teaching and learning. Evaluation begins with problem analysis which is an important first step in the development and evaluation of learning because the goals and barriers to learning are clarified in this step. Davis argues that ARABIYAT, ISSN: 2356-153X, E-ISSN: 2442-9473 276 Arabiyât Jurnal Pendidikan Bahasa Arab dan Kebahasaaraban, 6(2), 2019 evaluation is a simple process of giving or assigning values to a number of goals, activities, decisions, performance, processes, people, objects, and many others.14 evaluation is a simple process of giving or assigning values to a number of goals, activities, decisions, performance, processes, people, objects, and many others.14 Outcomes assessment is crucially important if study abroad and international experiences are to find a firm foothold in the curriculum and if curricular designers are to make wise decisions that earn the support of the executive leadership of the campus. Research needs to determine how [international service-learning] contributes to a student's readiness and preparedness to learn after returning to the home campus.15 Based on the above limitations, it can be concluded that evaluation, in general, can be interpreted as a series of activities systematically to determine something value (goals, activities, decisions, performance, and others) based on certain criteria through measurement or assessment. b. Functions and Objectives of Learning Outcomes Arabiyât Jurnal Pendidikan Bahasa Arab dan Kebahasaaraban, 6(2), 2019 Suparman formulated the steps in sequence as follows: 1) determine the purpose of the test, 2) make a specification table for each test (question grid), 3) write test items, 4) assemble the test, 5) write instructions for students about writing the answer and the time needed for each type of test, 6) write the answer key for each test item, 7) test the test, 8) analyze the results of the test, and 9) revise the test.17 Based on the description above, it can be understood that the procedures that must be taken in compiling evaluation tools are: 1) determining the information needed, 2) describing the learning objectives according to the defined learning objectives, 3) creating a specification table consisting of a list of behaviors, behavioral weights , the type of test, and the weight of the test, 4) using the condition component in the learning objectives as the basis for compiling the assessment instrument, 5) writing the item by observing the material used, 6) classifying the types of tests and giving an explanation of how to do each type of test, and 7) determine the score for each type of question. 18 Imam Makruf, Strategi Pembelajaran bahasa Arab, (Semarang: Need's Press, 2009), 18. J y j g j ( y ) 20 MetinTimucin & Hogai Aryoubi, “Integrating Arts In EFL Curricula: A Focus on Language Listening Skills”, International Journal of Languages’ Education and Teaching, Vol. 4, No. 2, 2016, 234. Language skill's Arabic as other languages has four language skills. By using the word skill it is understood that the most basic aspects of language are communication tools, and skills are the most basic part when using language. The four majors include listening skill, speaking skill, reading skill, and writing skill.18 Atwi Suparman, Desain Pembelajaran, (Jakarta: Departemen Pendidikan dan Kebudayaan, 1991), c. Steps to Arrange an Assessment Tool According to Soekartawi, a measuring instrument is said to be of good quality if: 1) testing what is to be tested. In other words, the design of a measuring instrument must be relevant to which assessment function is desired, 2) consists of a series of good measuring instruments, namely a valid, relevant, specific, representative, and balanced measuring instrument.16 The sequence of steps in compiling an assessment tool according to Arikunto is as follows: 1) determine the purpose of conducting a test, 2) make restrictions on the material to be used as a test, 3) formulate specific instructional objectives of each part of the material, 4) line all indicators in the preparation table also includes the behavioral aspects contained in the indicator, 5) compiling a specification table that contains the subject matter, the aspect of thinking that is measured along with the balance between the two things. 6) write down the items, based on the indicators already written in the indicator table and the aspects of behavior that are covered. In making a measuring instrument for the sake of evaluating learning outcomes, a certain procedure must be taken to obtain a good measuring instrument. 277 ARABIYAT, ISSN: 2356-153X, E-ISSN: 2442-9473 277 19 Juwariyah Dahlan, Metode Belajar Mengajar Bahasa Arab, (Surabaya: Al Ikhlas, Cet. I, 1992), 11 73. Arabiyât Jurnal Pendidikan Bahasa Arab dan Kebahasaaraban, 6(2), 2019 b. Speaking Skill 23 Noor Anida Binti Awang, “Enhancing Arabic Speaking Skills among Malay Students through Group Work Activities”, International Journal of Humanities and Social Science, Vol. 3, No. 21, 2013, 213. a. Listening Skill Listening is the skill of capturing and producing language acquired by hearing. In listening, it usually uses the direct method. This method rule is the initial lesson given by listening or listening training exercises, then followed by exercises saying sounds first, then short words, and finally longer sentences. The sentences are then combined into conversations and stories. The subject matter is written in phonetic notation, not spelling as is usually the case for grammar taught inductively, with composing lessons consisting of reproduction, from those who have been heard and spoken.19 Listening is an active process requiring participation on the part of the listener. Listening is a vital skill to acquire pronunciation, vocabulary, word stress, and syntax. Also, the comprehension of messages can be understood through the tone of voice, accent, and pitch.20 73. ARABIYAT, ISSN: 2356-153X, E-ISSN: 2442-9473 278 22 Salih Mahgoub EL-Tingari, “Strategies for Learning Second Language Skills: Arabic Speaking Skills in the Malaysian Context”, International Journal of English Language Teaching and Linguistics, Vol. 1, No. 1, 2016, 22. 21 Ahmad Fuad Effendy, Metodologi Pengajaran Bahasa Arab, (Yogyakarta : Pustaka Pelajar, 2004), 102. 21 Ahmad Fuad Effendy, Metodologi Pengajaran Bahasa Arab, (Yogyakarta : Pustaka Pelajar, 2004), 102. 22 Salih Mahgoub EL-Tingari, “Strategies for Learning Second Language Skills: Arabic Speaking Skills in the Malaysian Context”, International Journal of English Language Teaching and Linguistics, Vol. 1, No. 1, 2016, 22. 23 Noor Anida Binti Awang, “Enhancing Arabic Speaking Skills among Malay Students through Group Work Activities”, International Journal of Humanities and Social Science, Vol. 3, No. 21, 2013, 213. 24 Sri Mulatsih, Mursid Saleh, Warsono, Issy Yuliasri. “Ideational Meanings of Teachers’ Utterances in Reading and Writing Classes”, International Journal of Language Teaching and Education, , Vol. 2, No. 3, 20183, 275. 25 Ahmad Fuad Effendy, Metodologi Pengajaran Bahasa Arab, 140. c. Reading Skill Reading is considered as a receptive skill, while writing is a productive one. The way to teach these two skills should also be different. Reading is a complex cognitive process of understanding symbols to construct or derive meaning. When we read, we use our eyes to receive written symbols (letters, punctuation marks, and spaces) and we use our brain to convert them into words, sentences, and paragraphs that communicate something to us. Reading can be silent (in our head) or aloud (so that other people can hear). As mentioned above, reading is a receptive skill-through reading we can receive some information.24 d. Writing Skill Writing is a language skill that is used to communicate indirectly or not face to face with other people. What is meant by writing ability is the skill of making letters (large and small) by copying or imitating writings in the sentence structure. Writing skills that are more important are the ability to write based on the understanding of composition or the ability to compose a language/fabricate. As with reading, writing skills have two aspects, but in different relationships. First, skills form letters and master spelling. Second, proficiency gives birth to thoughts and feelings with writing.25 b. Speaking Skill Speaking is the ability to communicate directly in the form of conversation or dialogue. In general, the purpose of speaking exercises for beginner and intermediate levels is so that students can communicate verbally in simple Arabic. The stages of speaking practice are as follows: In the initial stages, speaking exercises can be said to be similar to listening practice. As stated earlier, in the listening exercise there is a stage of listening and imitating. The ultimate goal of listening practice is the ability to understand what is being listened to. While the final goal of pronunciation practice is the ability of expression, which is using ideas/thoughts/ messages to others. Both are absolute conditions for reciprocally effective oral communication.21 Every linguistic skill has certain targets that will differentiate it from other skills, the targets of speaking are as follows: 1) The learners should articulate the different Arabic sounds, and perform the different types of stress and patterns, in a correct manner. 1) The learners should articulate the different Arabic sounds, and perform the different types of stress and patterns, in a correct manner. 2) To be able to articulate the approximate and similar sound 3) To know the difference between long and short vowels 4) To express his/her ideas using the correct system of constructing a phrase in Arabic. 5) To acquire linguistic affluence that is equivalent to his/her age, and his/her standard of maturity and ability. 6) To understand some forms of Arabian culture. 7) To be able to think and communicate in Arabic for an acceptable duration of time.22 7) To be able to think and communicate in Arabic for an acceptable duration of time.22 Macaro stressed that effective speakers do not give up or hesitate for too long when they cannot think of how to say something. The researchers believe that to be a good speaker in the Arabic language, students must think positively no matter what others say about them. On the other hand, they must take part in tasks and activities that may encourage them to speak. Additionally, students must be more independent when they are at university.23 ARABIYAT, ISSN: 2356-153X, E-ISSN: 2442-9473 279 Arabiyât Jurnal Pendidikan Bahasa Arab dan Kebahasaaraban, 6(2), 2019 data from written sources, both collected by the lecturers concerned and the departments and faculties. data from written sources, both collected by the lecturers concerned and the departments and faculties. The research subjects in the context of this case research were lecturers who taught listening and reading courses for the 2017/2018 academic year, totaling 4 lecturers. The number of subjects of this study is used to learn and understand something about the selected case without the need to generalize to all cases. In determining the informants used snowball techniques, but this pattern is not applied in its entirety, because in certain circumstances according to the objectives to be achieved requires researchers determine themselves the next informant who will be found without first determined or notified by previous informants. Data collection techniques in this study were conducted in three ways, namely: in-depth interviews, non-participant observation, and documentation. The analysis during data collection in the field is done by 1) checking data, 2) organizing data, 3) coding data. While the analysis after the data has been collected is done through three paths, namely: 1) data reduction, 2) data presentation and 3) conclusion/verification.26 For the validity of the data, the author uses triangulation with the source that is comparing and checking the degree of trust or information obtained through time and different tools in qualitative research. Result and Discussion Performance of lecturers in designing learning language proficiency courses 26 Michael Quinn Patton, Qualitatife Evaluation Methods, (Beverly Hill : Sage Publication, 1987), 331. Method This research was conducted with a qualitative approach. This research was designed using a case study design, in the form of a study of lecturers who performed their performance. The types of data collected are related to 1) lecturer performance in designing learning which includes: subject identity, learning objectives, subject matter, determination of lecture time, information on learning strategies in class, reference books and other learning resources, 2) lecturers' performance in implementing learning, which includes: delivering outlines of lectures, presenting information including the methods used, establishing educational relations between lecturers and students, using learning media, closing learning activities, 3) lecturers' performance in evaluating learning outcomes which include: planning the determination of things related to student assignment assessment, compiling assessment instruments, designing the middle test, final test, and containing an explanation of the accumulation of all aspects that are used as elements of assessment in order to determine the final value college student. The data sources in this study were listening and reading lecturers, PBA department heads, and colleagues from PBA lecturers. According to Moleong, the main data sources in qualitative research are words and actions, the rest is additional ARABIYAT, ISSN: 2356-153X, E-ISSN: 2442-9473 280 Michael Quinn Patton, Qualitatife Evaluation Methods, (Beverly Hill : Sage Publication, 1987), b. Reading Course In designing reading skills learning courses namely reading, lecturer of the course he said that before the implementation of the course learning activities reading for one semester, he has made learning design in the form of SAP that he made based on the knowledge that he mastered, and already done before, even the design of the course is basically the same as the previous one, only updated about the lecture schedule. and he is always guided by the curriculum in the Arabic Language Education Department of the UIN STS Jambi, particularly regarding the standards of competence, basic competencies, and subject matter. Because this has been stipulated in the faculty curriculum so that lecturers are not given the authority to change. To direct the learning process, FW practices the arrangement of the material contained in SAP, each meeting is discussed one or two materials according to the time available. Learning material contained in SAP, in the form of subjects in accordance with the curriculum in the PBA department, and for more detailed elaboration contained in the teaching material that was set though not perfect. While the test for each subject matter according to FW he never fit in the design of learning, he only did it when the learning process took place, and for assessment FW took from several components, namely from the results of the assignments given, from mid semester exams, final semester exams, and from the presence of students following the lecture process. a. Listening Course In designing the subject course, the lecturer of the course has made SAP the same course he took care of in this semester, but he said he did not know whether the other lecturers who taught the same subjects also made SAP, to know that those who teach eyes This lecture is two lecturers, He said that: "Before I teach, I first made SAP, this is important I do as a guideline in implementing listening learning," but I admit that in making this SAP I did not coordinate with other lecturers. In making this SAP I do it myself and of course, based on my ability ". Furthermore, the caregiver of the course is not one hundred percent guided by the curriculum in the faculty, there are some things that are adopted according to the ability of students in the classroom, one of which is a material problem. Some of the topics of discussion he adopted from the curriculum that he considered were in accordance with the abilities and needs of students. Based on the SAP istima document, some of the components contained in the syllabus are as follows: course identity, basic competencies, the main material, and a description of the main material, learning experience, indicators, assessment, media, and teaching tools, and references. (documentation of listening) 331. ARABIYAT, ISSN: 2356-153X, E-ISSN: 2442-9473 281 Arabiyât Jurnal Pendidikan Bahasa Arab dan Kebahasaaraban, 6(2), 2019 from beginner to a high level are very easy to obtain and are provided by the department. Even so, it does not mean that audiovisual is not used at all, I still use VCD even though sometimes the content is not in accordance with syllabus but it is more interlude or entertainment so students are not saturated. from beginner to a high level are very easy to obtain and are provided by the department. Even so, it does not mean that audiovisual is not used at all, I still use VCD even though sometimes the content is not in accordance with syllabus but it is more interlude or entertainment so students are not saturated. At the end of the learning process listening usually provides an explanation of the material discussed especially if students feel many difficulties, both in the form of understanding vocabulary, understanding texts that are heard or difficulties in summarizing the main ideas. Among the efforts made by Mr. KU to motivate his students to practice listening to Arabic is always to advise students to play Arabic videos, whether in the form of videos of songs, chanting, drama or others. And sometimes Mr. KU gives video files to students to see in their homes. The first observation of the learning process maharatul istima ', Mr. MT entered the class by saying hello, then asked whether this student was really a participant in the maharatul istima ’course he was teaching. After most of the students answered "yes", then he began to explain about the SAP he had made after he briefly introduced himself and attended his students who at that time from 27 students written on the attendance list only 24 students attended, three people did not enter without information, this activity lasted 55 minutes. In explaining SAP, he began by mentioning the competencies that must be achieved by each student in this mahima ul istima 'course. After he explains the competencies and indicators, the next is, material, learning experience, assessment, and tools and sources of teaching. In the second observation, Mr. MT entered by saying greetings and then attending the student by name one by one. After completion, Mr. MT invited the students to get ready to listen to a simple reading text that he would read and then students were asked to record vocabulary that had no known meaning. Mr. a. Listening Course The implementation of istima 'learning courses carried out by Mr. KU in one semester ranges from 14 times face to face. To know the realization, it can be seen from his statements as follows: I usually carry out the learning process in this course by using the tadribat istimaiyah method, as well as lecture and question and answer methods. The tadribat istimaiyah method is a method of training students in listening to simple Arabic texts, then students are asked to identify and differentiate the types of letters that are close together, give meaning to a particular vocabulary, understand the contents of the message, and express the contents of the message in the form of spoken language and writing. While the lecture method I used in the first meeting was when introducing myself as needed while doing student attendance, followed by informing SAP. The question and answer method are carried out on the sidelines of the lecture method and tadribat istimaiyah. Learning media used in the istima learning process are language laboratories, tape recorders, cassettes, DVD players, laptops, infocus, blackboards, and board markers. Of the many media both audio and audiovisual, KU's father uses audio more often, namely tape and cassette than audio-visual, this is what he said: "I often use audio rather than audio visual because audio (tape) is very compatible with istima material 'also because equipment that helps such as materials and VCD is rather difficult to obtain when compared to cassettes. If the tapes to train students' hearing ARABIYAT, ISSN: 2356-153X, E-ISSN: 2442-9473 282 Arabiyât Jurnal Pendidikan Bahasa Arab dan Kebahasaaraban, 6(2), 2019 discussed and students are given questions to measure their understanding of the paragraph, and so on until it's finished. discussed and students are given questions to measure their understanding of the paragraph, and so on until it's finished. b. Subjects of Reading The implementation of reading learning conducted by Mr. FW in one semester ranges from 16 times face to face including in mid-semester and final semester examinations. To find out the realization can be seen from the statement and the description as follows: the teaching process that I did use the qira'ah method, assignment and practice directly in the classroom, and the question and answer method. In more detail, he stated that in the first meeting, besides informing about the objectives of the course, learning methods, materials, and methods of assessment, he also conducted a pre-test. This was done to measure the initial abilities of students so that I would not provide material that was far from their abilities for both low and high. Also as a measure of the progress, they achieved after the lecture was completed at the end of the semester. The media used by Mr. FW in the learning process of the Maharah al-Qira'ah course is commonly used tools such as infocus, blackboard, markers, books related to qira'ah sya'iah, and Arabic-Indonesian dictionaries. Observing the author in the field, Mr. FW entered the class 10 minutes late, then he said his greetings and then delivered the material to be discussed at this meeting as he had delivered at the previous meeting. Then he asked the students whether they had copied the qiraah material he had submitted last week, and it appeared that all students answered "yes already". Furthermore, Mr. FW wrote the theme that will be discussed on the board, to the students, they were asked to open their respective potencies, then Mr. FW told one by one the students to read the qiraah material in a religious manner about 20 minutes. Arabiyât Jurnal Pendidikan Bahasa Arab dan Kebahasaaraban, 6(2), 2019 MT read a simple reading text about a'malul yaumiiyah, all students watched him attentively and occasionally they wrote vocabulary that they did not know, MT repeated the reading for the second. After that, let the students ask for the vocabulary that they have not understood before giving questions about the differences in the sounds of the letters that are close together and about understanding the contents of the text that he read earlier. There were three students who asked about the meaning of the word and answered MT briefly without any lengthy explanation. The third observation, at this meeting, Mr. MT had prepared a video to be played and played to students, the topic to be discussed at this meeting was at-tarwih fil Islam, a relatively long reading text and had more complex expressions than the previous meeting. After attending and instructing students to prepare it, MT began playing the video and explained a little that the material this time was relatively more difficult and the content of the text was relatively more complex, so at this stage pay close attention and note if there are mufradat that have not been understood, then MT played back the video, but this time it was not played in its entirety but each one paragraph while ARABIYAT, ISSN: 2356-153X, E-ISSN: 2442-9473 283 b. Subjects of Reading Mr. FW stated that the evaluation of student learning outcomes in this muthalaah course was carried out in accordance with the evaluation requirements of the faculty as he said: "The four components that I usually use in determining the final grades of students in this learning, namely the collection of assignments during lectures, mid-semester exam scores, final semester exam scores, and participation values in accordance with applicable provisions that refer to ISO 9001 standards. In preparing the implementation of the assessment of the component, Mr. FW did it as follows: a) for attendance value including the participation of Mr. FW always gave a sign to each student who entered and did not enter for several reasons. b) for mid-semester exam preparation, Mr. FW does not make item questions in detail, because the exam is carried out verbally, the preparation is only with attendance and syllabus courses, so questions are developed by FW when in the classroom.c) for exam questions at the end of the semester, Mr. FW gave assignments to students and was collected during the exam schedule for the course. d) for tasks already prepared in advance, namely sometimes taken from several references, or sometimes he himself made it. a. listening Course In evaluating, Mr. KU stated that the evaluation of student learning outcomes in this istima 'course was carried out in accordance with the evaluation rules and regulations of the Department of Arabic Language Education, Tarbiyah Faculty, UIN STS Jambi. In his interview he told the author as follows: the components that I used in the assessment in determining the final grades of students apart from the mid- semester grades and the end of semester grades, as well as assignments. For the mid-semester exam and the end of the semester Mr. KU said: I scheduled the mid-semester exam at the eighth meeting held in the labor language room with the material they had studied, with 10 questions, while I always carried out the semester exams outside the exam schedule determined by the faculty, this is considering the examination in the language laboratory room, and I schedule one week before the final semester exam begins. ARABIYAT, ISSN: 2356-153X, E-ISSN: 2442-9473 284 284 Arabiyât Jurnal Pendidikan Bahasa Arab dan Kebahasaaraban, 6(2), 2019 The provision of whether or not students take part in the final semester exams depends on their attendance in the lecture process, if their attendance is less than 80% in one semester, then the person may not take the final exam, except if the person meets the lecturer and explains the reason for his absence and the lecturer can accept the reason if not, it means that the person concerned is not entitled to take the final examination, so the possibility of not passing is very large. Submitting the final grade to the academic section conducted by KU is always on time for the submission deadline, this is done so that there is no same value given by the academic section, because it is in accordance with the circular letter from the dean 1, that if the lecturers set the deadline do not submit a list of grades of students, the value will be taken over by the department, and given sanctions not guiding and testing the thesis and compensation in the following semester. b. Subjects of Reading Arabiyât Jurnal Pendidikan Bahasa Arab dan Kebahasaaraban, 6(2), 2019 delivering conclusions from the material that has been discussed, 3) the evaluation of learning outcomes is an activity of measuring the level of success and ability of students in the learning process.[] delivering conclusions from the material that has been discussed, 3) the evaluation of learning outcomes is an activity of measuring the level of success and ability of students in the learning process.[] Conclusion Performance of Arabic education lecturers faculty of Tarbiyah UIN STS Jambi in learning language devotion that lecturer have not maximized the potential in giving lectures, coupled with the preparation of SAP courses that are lacking this is seen in 1) the design of learning made by lecturers listening and reading in the form of SAP, only briefly explains about several components, namely the identity of the course, learning outcome indicators, lecture method, assessment, and bibliography, mentions several reading sources used in the listening learning process and reading, 2) the implementation of learning include preliminary activities in the form of mentioning lecture material to be studied in one semester, core activities by applying patterns learning that further activate students to practice expressing their ideas in writing and understanding the texts that are played. And closing activities, in the form of ARABIYAT, ISSN: 2356-153X, E-ISSN: 2442-9473 285 REFERENCES Anida, Noor. “Enhancing Arabic Speaking Skills among Malay Students through Group Work Activities”, International Journal of Humanities and Social Science, Vol. 3, No. 21, 2013. Arikunto, Suharsimi. Dasar-Dasar Evaluasi Pendidikan. Jakarta: Bumi Aksara, 2015. Atwi, Suparman. Desain Pembelajaran. Jakarta: Departemen Pendidikan dan Kebudayaan, 1991. Azmahani Aziza, Azmahani., Khairiyah M. Yusof., Jamaludin M. Yatim. “Evaluation on the Effectiveness of Learning Outcomes from Students’ Perspectives”, International Conference on Teaching and Learning in Higher Education (ICTLHE), in conjunction with RCEE & RHED, 2012. Conole, Karen Fill Gráinne. “A learning design toolkit to create pedagogically effective learning activities”, Journal of Interactive Media in Education, Vol. 8, 2005. Dahlan, Juwairiyah. Metode Belajar Mengajar Bahasa Arab. Surabaya: Al-Ikhlas, t.t, Cet I. Davis, Wor. K. 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https://openalex.org/W4254528509	https://bmcpublichealth.biomedcentral.com/track/pdf/10.1186/s12889-020-09839-3	English	null	Serious non-fatal unintentional injuries among in-school adolescents in Sri Lanka: results from the 2016 Sri Lankan Global School-Based Health Survey	Research Square (Research Square)	2,020	cc-by	6,492	Wickramasinghe et al. BMC Public Health (2020) 20:1697 https://doi.org/10.1186/s12889-020-09839-3 Wickramasinghe et al. BMC Public Health (2020) 20:1697 https://doi.org/10.1186/s12889-020-09839-3 Open Access Serious non-fatal unintentional injuries among in-school adolescents in Sri Lanka: results from the 2016 Sri Lankan global school-based health survey Sashimali Wickramasinghe1* , Nalika Sepali Gunawardena2, Dhanusha Punyadasa1, Shanthi Gunawardena1, Champika Wickramasinghe1, Ayesha Lokubalasooriya1, Renuka Peiris3 and Sameera Senanayake4 * Correspondence: sashimali2004@yahoo.com 1Ministry of Health, Colombo, Sri Lanka Full list of author information is available at the end of the article * Correspondence: sashimali2004@yahoo.com 1Ministry of Health, Colombo, Sri Lanka Full list of author information is available at th © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Abstract Background: Unintentional injuries among adolescents is a major public health problem the world over. A great majority of the annual deaths among adolescents is due to unintentional injuries; it is also the leading cause of death among adolescents in the world. The aim of this study was to estimate the incidence of injuries and their associated factors among school going adolescents aged 13–17 years using data from the most recent Global School-Based Health Survey (GSHS) conducted in Sri Lanka. Methods: A cross-sectional survey was conducted using a self-administered questionnaire, among 3262 adolescents attending government schools. The sample was selected through a two-staged cluster sampling technique. In the first stage, 40 schools were selected using probabilities proportional to school enrollment size, from all schools in the country that have classes in grades 8–12. Then, from the selected schools, classes were selected using systematic equal probability sampling with a random start. The weighted prevalence was calculated, and logistic regression analysis was conducted in order to determine the correlates. Results: During the 12 months before the survey, 35.8% (95% CI-30.7 - 41.1) of the students reported being seriously injured one or more times. The injuries were more common among males, but were equally common among the two different age groups (13–15 age group vs 16–17 age group). The most common type of injury was cut or stab wounds (5.5%), followed by broken bones/dislocated joints (5.3%). Multivariable analysis revealed that only a few factors were associated with injury, such as being of the male sex, being bullied, being physically attacked, and/or being in a physical fight. Conclusion: This study demonstrated that the prevalence of serious unintentional injuries among school going adolescents is a major public health problem in Sri Lanka. This timely and comprehensive survey would help policy makers and researchers identify the unmet needs related to adolescent injuries. Furthermore, evidence generated form the study should be given due consideration when designing school-based interventions to prevent adolescent injuries. Keywords: Unintentional injuries, Adolescents, Sri Lanka * Correspondence: sashimali2004@yahoo.com 1Ministry of Health, Colombo, Sri Lanka Full list of author information is available at the end of the article Background Prevention (CDC), is a rich source of information on health and related information on school going adoles- cents [11]. g Adolescents are vulnerable to multifarious injuries be- cause of their size, growth and development, inexperi- ence, and natural curiosity [1]. These injuries can occur either intentionally or unintentionally. Intentionality dis- tinguishes violence from unintended events that result in injury [1]. Unintentional injuries among adolescents is a major public health crisis the world over. Interestingly, more than 95% of injury related deaths among adoles- cents occur in low and middle-income countries (LMIC) [2]. Analysis of data from 68 LMICs indicated that the overall prevalence of unintentional injuries among ado- lescents in the 13–15 year age group during the past 12 months is 42.9% (95% CI 39.0–46.9) [3]. Furthermore, the leading cause of death among adolescents aged 15– 19 years is unintentional injuries [4]. Adolescents consist of one fifth of the population of Sri Lanka. Among the estimated number of 3.8 million adolescents in the country, close to 70% of adolescents attend school [12]. The GSHS was first conducted in Sri Lanka in 2008 among school going adolescents, and it provided valuable information on their health behaviors. This study revealed that among students in the 13–15 year age group, 37.2% (95% CI-32. 1–42.2) have had at least one serious injury in the previous year. Obtaining a clear view of the burden of the issue necessitates detailed information on the magnitude, trends, and influencing factors of the problem. Furthermore, repeated GSHSs are recommended by the WHO to assess the trends in the prevalence of risk factors [13]. Evidence indicates that for every injured adolescent who dies, there are several thousand adolescents who live on with varying degrees of disability [5]. Further- more, significant economic costs and lifelong social bur- dens are caused by non-fatal adolescent injuries [6]. Considering the type of unintentional injuries, the five most common injuries in adolescents are road traffic in- juries, falls, burns, drowning, and poisoning [7, 8]. These five categories make up 60% of deaths due to adolescent injury worldwide [2]. Other injuries that could occur are open wound injuries, fractures, and muscle and joint in- juries [9]. Thus, the aim of this paper is to estimate the incidence of injuries and their associated factors among school go- ing adolescents aged 13–17 years using the data of the most recent GSHS conducted in Sri Lanka. © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Wickramasinghe et al. BMC Public Health (2020) 20:1697 Page 2 of 8 Wickramasinghe et al. BMC Public Health (2020) 20:1697 Page 2 of 8 Background This is the first national survey conducted among school going ado- lescents since the first GSHS conducted in 2008. There- fore, the information on the extent of adolescent unintentional injuries and associated factors in local set- tings gathered from this survey would be useful for health policy makers in the formulation of injury preven- tion policies and a strategic framework. It could also be useful for integrating injury preventive interventions into the existing school health programme in Sri Lanka. In developed countries, unintentional injuries have re- placed infectious diseases as the most serious public health problem among adolescents [1, 10]. However, the true extent of the problem in developing countries is largely unknown owing to the lack of national level sur- veys. Therefore, information is urgently needed on the incidence rates as well as the factors contributing to adolescent unintentional injuries in developing countries. Methods This descriptive cross-sectional study involved the ana- lysis of existing data from the Sri Lankan Global School- Based Health Survey conducted in 2016. The GSHS in- tends to provide data on health behaviors and associated risk and protective factors among school going adoles- cents. Students of grades 8, 9, 10, 11 and 12 in govern- ment schools (which corresponds to the 13 to 17 year age group) in Sri Lanka were recruited for the survey. Data collection was done during the period 1st October 2016 to 31st November 2016. The methodology devel- oped by the WHO and the US Center for Disease Con- trol and Prevention (CDC) has been adopted in the current study [14]. Similar to many developing countries, there is a scarcity of knowledge in this regard in Sri Lanka. Most studies on adolescent injuries conducted in Sri Lanka are hospital- based studies. Injuries necessitating hospitalization are relatively small in number, and hospital data is only the tip of the iceberg [2]. Therefore, national level community-based studies are essential to identify the true magnitude of the problem in addition to prioritizing pre- ventive strategies. W ¼ W1W2f 1f 2f 3 W ¼ W1W2f 1f 2f 3 W1 = Inverse probability of the school being selected. W2 = Inverse probability of the classroom being se- lected within the school. The questionnaire f3 = Adjustment factor estimated by grade, after post- stratification. Incidences of injuries are presented in terms of having sustained serious injuries, and were reported using fre- quencies with 95% confidence intervals. The correlates of sustaining a serious injury were identified by conduct- ing a bivariate analysis and a backward logistic regres- sion analysis. The dependent variable was sustaining a serious injury within 12 months prior to the survey at least once or not. The independent variables were all categorical in type and included age, sex, being bullied, considering suicide, attempting suicide, feeling lonely most of the time, current substance use (alcohol, ciga- rettes, recreational drugs etc.), not having close friends, missing classes without permission, parents not under- standing students’ problems, parents not knowing what students were doing during their free time, not being taught anger management in school, being in a physical fight, and being physically attacked. Except for age and sex, all other variables had more than one response and were dichotomized by categorizing any positive response as “Yes” and any negative response as “No”. Variables that resulted in a p value of 0.05 or less in the bivariate analysis were included in the backward multivariable lo- gistic regression. Adjusted odds ratios with 95% confi- dence intervals were obtained for the significant associates identified by the logistic regression. Results Among the 3650 students who were sampled, 3263 (89%) completed the questionnaires. However, only 3262 questionnaires were usable for the final analysis. Of the sampled population, 387 (3650 minus 3263) did not par- ticipate in the study. The reasons included not giving consent (n = 116) and being absent from school on the day of data collection (n = 271). Relevant administrative clearance to conduct the study was obtained from the Ministry of Education, zonal edu- cation directors, and school principals. Ethical clearance to conduct the study was received from the Faculty of Medicine, University of Colombo. Sample size and sampling The Global School-Based Student Health Survey (GSHS), which was initiated in 2001 as a collaborative effort of the World Health Organization (WHO), The United Nations Joint Programme on HIV/AIDS (UNAIDS), The United Nations Educational, Scientific and Cultural Organization (UNESCO), and the United Nations Children’s Fund (UNICEF) along with technical assistance from the US Center for Disease Control and A sample of 3125 students was deemed adequate to as- sess the magnitude of injuries in this group with a preci- sion of ±5% and an 80% response rate. The sample was selected through a two-stage cluster sampling technique. In the first stage, 40 schools were selected, using prob- abilities proportional to school enrollment size, from all schools in the country that have grades 8–12 classes. Page 3 of 8 Wickramasinghe et al. BMC Public Health (2020) 20:1697 Page 3 of 8 Page 3 of 8 Wickramasinghe et al. BMC Public Health (2020) 20:1697 Then, from the selected schools, classes were selected using systematic equal probability sampling with a ran- dom start. All students in the selected classes were in- vited to participate in the survey. The questionnaire f1 = Non-response adjustment factor (i.e. school level) estimated based on the school size (small, medium, large). The GSHS questionnaire was adapted to Sri Lanka and was translated into Sinhala and Tamil. This was a self- administered questionnaire. Three questions were in- cluded in the questionnaire to obtain information rele- vant to injuries. The first question assessed the number of times the student was seriously injured during the past 12 months; here, seriousness was defined as ‘making the respondent miss at least one full day of usual activ- ities (such as school, sports, or a job) or necessitating treatment by a doctor or nurse’. It had eight responses, with the frequency ranging from 0 to 12 or more times. The next question inquired about the most serious in- jury that had happened to the respondent during the 12 months prior to the survey. The measurement scale had the following responses: I was not seriously injured dur- ing the past 12 months; I had a broken bone or a dislo- cated joint; I had a cut or stab wound; I had a concussion or other head or neck injury, was knocked out, or could not breathe; I had a gunshot wound; I had a bad burn; I was poisoned or took too much of a drug; Something else happened to me. The final question looked into the cause of the most serious injury that had happened during the past 12 months. The responses in- cluded; I was not seriously injured during the past 12 months; I was involved in a motor vehicle accident or hit by a motor vehicle; I fell; Something fell on me or hit me; I was attacked or abused or was fighting with some- one; I was in a fire or too near a flame or something hot; I inhaled or swallowed something bad for me; Some- thing else caused my injury. Information required to as- sess the associations (socio demographic details, details of students’ mental health, substance use, and parental engagement in students’ life) were collected. Measures were taken to minimize any interference with school ac- tivities during data collection. g f2 = Non-response adjustment factor (i.e. student level) estimated by class. f2 = Non-response adjustment factor (i.e. student level) estimated by class. f2 = Non-response adjustment factor (i.e. student level) estimated by class. f3 = Adjustment factor estimated by grade, after post- stratification. Characteristics of the study sample The SPSS version 21.0 was used for data analysis. The data set was cleaned and edited for inconsistencies. Missing data were not statistically imputed. The follow- ing weight (W) was assigned to each questionnaire in order to allow for differing patterns of non-response and the likelihood of sampling each student: The majority of respondents were 13–15 years old (66.5%,), and females comprised 51.1% of the sampled population (Table 1). During the 12 months before the survey, 35.6% (95% CI-30.7 - 41.1) of the students reported being seriously injured one or more times. Almost one out of two boys Wickramasinghe et al. BMC Public Health (2020) 20:1697 Page 4 of 8 Table 1 Description of study participants of the 2016 Sri Lankan Global School-Based Survey (including the variables used to assess the correlates of unintended injuries) Characteristic Numbera Percentageb Sex (n = 3262) Male 1437 48.9 Female 1805 51.1 Age (n = 3261) < 13 years 66 2.1 13–15 years 2196 66.5 16–17 years 977 30.7 > 17 years 22 0.7 Was Bullied (n = 3196; missing =66) 1208 38.6 Felt lonely most of the time (n = 3250; missing =12) 294 9.1 Considered suicide (n = 3221; missing =41) 298 9.4 Current smoking (n = 3244; missing =18) 107 3.6 Current use of alcohol (n = 3191; missing =71) 103 3.3 Current use of recreational drugs (n = 3217; missing =45) 79 2.7 Had no close friends (n = 3245; missing =17) 180 5.6 Lacked parental supervision (n = 3233; missing =29) 987 30.8 aUnweighted frequency; bWeighted percentage that falls were the most common mechanism (12.1%), followed by motor vehicle crashes (3.0%) and something falling on the person (2.7%). Falls were the most com- mon mechanism for injuries among both age groups and both sexes (Table 4). (45.3, 95% CI 39.7–51.1) had been seriously injured as compared to only one fourth of girls (26.4%), which was statistically significant. Serious injuries were equally common among students in younger (13–15 years) age groups and older (16–17 years) age groups (p > 0.05) (Table 2). Discussion injured during the past 12 months. Similar to other LMIC countries, our study indicates that adolescent non-fatal injuries have the capacity to affect the daily ac- tivities of school going adolescents. Adolescents are more prone to injuries due to risk tak- ing and sensation seeking behaviors [15]. This first na- tional survey conducted among school going adolescents after the GSHS-2008 indicated that the common finding in the study group includes serious injuries necessitating medical attention and missing out daily activities. Ap- proximately one in three children in the 13–17 year age group was seriously injured at least once within the 12 months prior to the survey. The prevalence of uninten- tional injuries has not changed over the years. A similar prevalence (37.2%) was found in the GSHS-2008 among 13–15 year olds [16], which indicates the necessity for novel public health approaches to combat this serious menace. The current study was conducted as a community- based study, which is able to paint a more comprehen- sive picture of injuries when compared to hospital based studies [17, 18]. This is best described as an injury pyra- mid based on the level of medical treatment of injury victims [19]. The apex of the pyramid represents the in- juries necessitating hospitalization, which are relatively small in number, whereas lower parts of the pyramid represent the more numerous injuries, which are of lesser severity. Since hospital-based studies can identify injuries that required hospitalization, they will only ad- dress the apex of the injury pyramid, which is the tip of the iceberg. Therefore, it is imperative to conduct community-based studies on adolescents’ unintentional injuries to identify the true magnitude of the problem. Unintentional injuries are a major contributor to ado- lescent mortality and morbidity. Direct comparison of results between countries and even within the same country, across different studies, is difficult due meth- odological differences (e.g. age of the study population). However, the comparison of GSHS studies provides use- ful information as they have used uniform definitions and study populations. Han et al. (2019) conducted a secondary analysis of GSHS data from 68 LMIC coun- tries. Factors associated with violence and injury The most common type of injury was a cut or stab wound (5.5%), followed by a broken bone/dislocated joint (5.3%) and a head injury or a concussion (1.2%). While the cut or stab wounds were the commonest in- jury type among the younger age group, broken bones/ dislocated joints were the commonest injury type among the older age group. Broken bones/ dislocated joints were the commonest injury type among males, while among females, the commonest injury type was cut or stab wounds (Table 3). Bivariate analysis revealed that being of the male sex, be- ing bullied, feeling lonely most of the time within the past 12 months, considering suicide, smoking, alcohol use, use of recreational drugs, being physically attacked or being in a physical fight, and lack of parental supervi- sion were significantly associated with being seriously in- jured at least once in the year prior to the survey. Multivariable analysis revealed that only a few factors were associated with injury, namely, being of the male sex, being bullied, being physically attacked, and/or be- ing in a physical fight (Table 5). Analysis of the mechanism of the most serious injury that had happened during the past 12 months revealed Table 2 Incidence of unintentional injuries among study participants (N = 3262) Table 2 Incidence of unintentional injuries among study participants (N = 3262) Was seriously injured at least once, within 12 months prior to survey % (95% CI) Total 35.6 (30.7–41.1) Sex Males 45.3 (39.7–51.1) Females 26.4 (21.6–32.2) Age 13–15 years 35.9 (30.3–41.9) 16–17 years 34.8 (29.1–40.9) Table 2 Incidence of unintentional injuries among study participants (N = 3262) Was seriously injured at least once, within 12 months prior to survey % (95% CI) Total 35.6 (30.7–41.1) Sex Males 45.3 (39.7–51.1) Females 26.4 (21.6–32.2) Age 13–15 years 35.9 (30.3–41.9) 16–17 years 34.8 (29.1–40.9) Wickramasinghe et al. Factors associated with violence and injury BMC Public Health (2020) 20:1697 Page 5 of 8 Page 5 of 8 Table 3 Description of the serious injuries that has happened to the study participants Type of injury Age group Sex Total (N = 3262) 13–15 years (N = 2196) 16–17 years (N = 977) Male (N = 1437) Female (N = 1805) N % N % N % N % N % Cut or stab wound 134 6.1 41 4.2 113 7.9 65 3.6 181 5.5 Broken bone/ dislocated joint 121 5.5 49 5.0 116 8.1 57 3.2 173 5.3 Concussion/ Head injury 34 1.5 6 0.6 22 1.5 16 0.9 40 1.2 Burns 19 0.9 1 0.1 7 0.5 13 0.7 22 0.7 Poisoned 5 0.2 4 0.4 6 0.4 6 0.3 12 0.4 Something else 229 10.4 139 14.2 205 14.3 176 9.8 381 11.7 Table 3 Description of the serious injuries that has happened to the study participants Discussion The overall incidence of serious injuries among 12–15 year old adolescents was 42.9% [3], which means that roughly one in two adolescents has been seriously A major limitation in comparing incidence data of un- intentional injuries across studies is the lack of consistency in their assessment, recall periods, and defi- nitions. It has been found that there is a wide variation in the incidence of injuries with varying recall periods. The results of studies using diverse recall periods cannot be compared without bearing in mind the factors affect- ing such differences [20]. However, since GSHSs have Table 4 Description of the serious injuries that has happened to the study participants T f i j A S Table 4 Description of the serious injuries that has happened to the study participants Type of injury Age group Sex Total (N = 3262) 13–15 years (N = 2196) 16–17 years (N = 977) Male (N = 1437) Female (N = 1805) N % N % N % N % N % Falls 293 13.3 95 9.7 223 15.5 168 9.3 395 12.1 Motor vehicle accidents 53 2.4 39 4.0 74 5.1 24 1.3 99 3.0 Something falling on the person 61 2.8 22 2.3 59 4.1 28 1.6 87 2.7 Attacks 14 0.6 8 0.8 13 0.9 9 0.5 22 0.7 Fire 7 0.3 1 0.1 6 0.4 3 0.2 9 0.3 Breathed a noxious material 8 0.4 3 0.3 4 0.3 9 0.5 14 0.4 Something else 119 5.4 66 6.8 91 6.3 97 5.4 190 5.8 Wickramasinghe et al. BMC Public Health (2020) 20:1697 Page 6 of 8 Table 5 Factors associated with being physically attacked (results of bi-variable and multi-variable analysis) Characteristic Bi-variable analysis OR (95%CI) Multi-variable analysis aOR (95%CI) Age 13–15 yrs 1.0 (0.8–1.2)* – 16–17 yrs 1 Sex Male 2.3 (1.9–2.7) 1.8 (1.5–2. 1) Female 1 1 Was Bullied Yes 4. 1 (3.5–4.8) 2.3 (1.9–2.5) No 1 1 Felt lonely Yes 2. 1 (1.6–2.7) – No 1 Considered suicide Yes 2.2 (1.7–2.8) – No 1 Current smoking Yes 3.8 (2.5–5.8) – No 1 Current use of alcohol Yes 3.2 (2.1–4.9) – No 1 Current use of recreational drugs Yes 7.7 (4.3–13.6) – No 1 Had close friends No 1.3 (0.9–1.8)* – Yes 1 Lacked parental supervision Yes 1.6 (1. 1–1.9) – No 1 Was physically attacked Yes 3.5 (3.0–4. Discussion 1) 1.6 (1.3–1.9) No 1 1 Was in a fight Yes 3.5 (3.0–4. 1) 1.9 (1.6–2.3) No 1 1 *Not significant at p = 0.05 level ble 5 Factors associated with being physically attacked (results of bi-variable and multi-variable analysis) been conducted in several countries and they have all used a consistent definition and a recall period, it en- ables meaningful comparison across countries. Analysis of GSHS data of 13 to 15 year old school students of Indonesia, Myanmar, Sri Lanka and Thailand for the purpose of identifying the burden of serious injuries among school students has reported an overall preva- lence of 42.2% for all countries, ranging from 27.0% in Myanmar to 46.8% in Thailand [21]. These figures indi- cate that the prevalence among low and middle-income countries is uniform across countries. However, rela- tively low figures have been reported from developed countries [9, 22]. been conducted in several countries and they have all used a consistent definition and a recall period, it en- ables meaningful comparison across countries. Analysis of GSHS data of 13 to 15 year old school students of Indonesia, Myanmar, Sri Lanka and Thailand for the purpose of identifying the burden of serious injuries among school students has reported an overall preva- lence of 42.2% for all countries, ranging from 27.0% in Myanmar to 46.8% in Thailand [21]. These figures indi- cate that the prevalence among low and middle-income countries is uniform across countries. However, rela- tively low figures have been reported from developed countries [9, 22]. males have a higher probability of risk taking behavior, are physically more active, and have more probability of engaging in competitive sports [25, 26]. Several studies have demonstrated that older adolescents are more prone to unintentional injuries [27, 28]. However, similar to the GSHS results of 2008, the present study has failed to demonstrate this association. The current study found that falls and motor vehicle accidents are the most common causes of unintentional injuries among school going adolescents, and similar studies have found these two to be the most common etiologies for the injuries [21, 29, 30]. Similarly, a large- scale household survey conducted to assess the epidemi- ology of unintentional injuries in Nepal has reported falls as the commonest cause for unintentional injuries among adolescents [31]. Likewise, vehicle accidents are also a leading cause of non-fatal injuries among adoles- cents [32]. Acknowledgements In addition to ill health, serious injuries also have dire effects on school performance. Thus, proper attention should be given to the provision of remedial measures for this problem. Knowledge about the extent and corre- lates of the problem is a valuable asset while designing remedial measures. According to the current study, be- ing of the male sex, being bullied, being physically attacked and/or being in a physical fight were associated with suffering serious injuries. Identification of such vul- nerable populations should be targeted while planning interventions for injury prevention. Authors would like to acknowledge Dr. Manju Rani (Regional Advisor - NCD and Tobacco Surveillance) and Mr. Naveen Agarwal of WHO SEARO, Delhi for technical and logistical support. Ms. Leanne Riley (WHO – Geneva), Dr. Laura Kann (CDC - Atlanta, USA) and others at CDC for providing technical support. All the students who have participated in the survey with enthusiasm, honesty and willingness. Funding This study has several limitations. Firstly, as the study was conducted among school going adolescents, general- izing the findings to the entire adolescent population in Sri Lanka is not possible, as the circumstances of the various adolescents may differ significantly. Secondly, the standard questionnaire used in the study did not have the geographical location of the injury. This could have been an important piece of knowledge with which to plan the intervention. Thirdly, the study population was school-going adolescents between 13 and 17 years, and it therefore does not represent the total school- going adolescent population, as, according to the WHO, an adolescent is defined as an individual between 10 and 19 years of age. Fourthly, the current study only included adolescents attending government schools, excluding those who attend private schools in the country. How- ever, since private school attendees comprise only 3% of the school going population [35], the authors believe that the current sample adequately represents the total school going population. Finally, recall bias is a possible limitation, as information was solicited regarding the 12 months prior to the survey, and participants may not have remembered the information accurately. This study was funded by the World Health Organization (WHO). The funder only provided financial assistance to conduct the study and had no role in the study design, data collection, data analysis, data interpretation, or writing the manuscript. Author details 1 1Ministry of Health, Colombo, Sri Lanka. 2World Health Organization, Country Office, Colombo, Sri Lanka. 3Ministry of Education, Pelawatta, Sri Lanka. 4Queensland University of Technology (QUT), Brisbane, Australia. 1Ministry of Health, Colombo, Sri Lanka. 2World Health Organization, Country Office, Colombo, Sri Lanka. 3Ministry of Education, Pelawatta, Sri Lanka. 4Queensland University of Technology (QUT), Brisbane, Australia. Received: 12 May 2020 Accepted: 4 November 2020 Authors’ contributions SW, SS, NSG, AL; Research idea, study design, analysis and interpretation. SS, SW, DP; Drafting of the manuscript. SW, SS, NSG, AL, SG, CW, RP, DP; Data analysis, interpretation, supervision and mentorship. All authors read and approved the manuscript. SW, SS, NSG, AL; Research idea, study design, analysis and interpretation. SS, SW, DP; Drafting of the manuscript. SW, SS, NSG, AL, SG, CW, RP, DP; Data analysis, interpretation, supervision and mentorship. All authors read and approved the manuscript. Availability of data and materials The dataset used during the current study is available from WHO s NCD Microdata Repository (https://extranet.who.int/ncdsmicrodata/index.php/ catalog/648). Discussion Evidence indicates that vehicle accidents among adolescents cause a sizeable economic burden due to loss of productivity and medical costs [33]. Fur- thermore, it has been shown that the commonest vehicle Boys are more prone to serious injuries that necessi- tate medical attention and/or lead to missing out daily activities, which fact was corroborated by the current study. The male gender was found to be significantly as- sociated with the occurrence of unintentional injuries in the present study. This was in line with some other stud- ies which had shown similar sex differences in injury rates [22–24]. This may be explained by the fact that Wickramasinghe et al. BMC Public Health (2020) 20:1697 Page 7 of 8 Page 7 of 8 a major public health problem in Sri Lanka. Health pol- icy and interventions related to the prevention of adoles- cent serious non-fatal injuries require solid evidence. This timely and comprehensive survey would help policy makers and researchers identify the unmet needs in this sphere. School-based interventions to address these is- sues should be designed with the goal of increasing ado- lescents’ awareness of possible preventive measures related to unintentional injuries. involved in these accidents is the motor cycle, and most of the injured children were not wearing helmets at the time of injury [29]. Riding motor cycles without a helmet is common among adolescents in Sri Lanka, which points to a serious public health problem among adoles- cents that needs urgent attention [34]. This study has also reported that fatal road traffic accidents are more commonly seen among boys than among girls. Though physical attacks have contributed to only 2.7% of all injuries, being in a fight was significantly associated with being injured. Globally, violence leads to 5.8% of in- jury related deaths among children below 17 years, and is more commonly seen among older adolescent groups (2). Therefore, more attention should be given to inter- personal violence among school children. Abbreviations CDC: US Centres for Disease Control and Prevention; GSHS: Global School- based Student Health Survey; UNAIDS: United Nations Joint Programme on HIV/AIDS; UNESCO: United Nations Educational, Scientific and Cultural Organization; UNICEF: United Nations Children’s Fund; WHO: World Health Organization; YLDs: Years Lost due to Disability Ethics approval and consent to participate The study is in accordance with Helsinki Declaration. The ethical clearance to conduct the study was received from Faculty of Medicine, University of Colombo, Sri Lanka (EC-16-184). 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https://openalex.org/W4206964671	https://edepot.wur.nl/559192	English	null	Cumulative risks from combined exposure to multiple pesticide residues in fruit and vegetables	null	2,021	cc-by-sa	13,976	WUR Biometris Rapport 47(12.21) https://doi.org/10.18174/559192 Project Kennis- en modelkoppelingen voor borging voedselveiligheid in de groenten en fruit sector Hilko van der Voet1, Johannes Kruisselbrink1, Marco van Lenthe1, Waldo de Boer1, Astrid Meewisse2 1 Wageningen University & Research, Biometris 2 Food Compass 1 Wageningen University & Research, Biometris Dit onderzoek is in opdracht van het Ministerie van LNV uitgevoerd door de Stichting Wageningen Research (WR), business unit Biometris, in het kader van beleidsondersteunend onderzoeksthema Voedselveiligheid (projectnummer TU18039, gunningscode BO-49-002-006). WR is een onderdeel van Wageningen University & Research, samenwerkingsverband tussen Wageningen University en de Stichting Wageningen Research. Wageningen, December 2021 WUR Biometris Rapport 47(12.21) https://doi.org/10.18174/559192 van der Voet, H., Kruisselbrink, J.W., van Lenthe, M., de Boer, W.J., Meewisse, A. (2021). Cumulative risks from combined exposure to multiple pesticide residues in fruit and vegetables. Wageningen University & Research Biometris report 47 (12.21). https://doi.org/10.18174/559192 van der Voet, H., Kruisselbrink, J.W., van Lenthe, M., de Boer, W.J., Meewisse, A. (2021). Cumulative risks from combined exposure to multiple pesticide residues in fruit and vegetables. Wageningen University & Research Biometris report 47 (12.21). https://doi.org/10.18174/559192 Keywords: Residues, pesticides, cumulative risk, software, MCRA, Food Compass © 2021 Wageningen, Sichting Wageningen Research, Business unit Biometris, Postbus 16, 6700 AA Wageningen; T 0317 48 40 85; www.wur.nl/biometris Copyright licentie: CC-BY-SA 4.0 © 2021 Wageningen, Sichting Wageningen Research, Business unit Biometris, Postbus 16, 6700 AA Wageningen; T 0317 48 40 85; www.wur.nl/biometris Copyright licentie: CC-BY-SA 4.0 © 2021 Wageningen, Sichting Wageningen Research, Business unit Biometris, Postbus 16, 6700 AA Wageningen; T 0317 48 40 85; www.wur.nl/biometris Copyright licentie: CC-BY-SA 4.0 KvK: 09098104 te Arnhem VAT NL no. 8113.83.696.B07 Stichting Wageningen Research is niet aansprakelijk voor eventuele schadelijke gevolgen die kunnen ontstaan bij gebruik van gegevens uit deze uitgave. Project Kennis- en modelkoppelingen voor borging voedselveiligheid in de groenten en fruit sector WUR Biometris Report 47 (12.21) 3 3 Contents Summary 5 1 Introduction 6 1.1 Overview and aim of the study 6 1.2 Development of a web portal to link private and public data 6 2 Data 8 2.1 Monitoring data (Food Compass) 8 2.2 Catalogues of substances and food products (GroentenFruit Huis) 8 2.2.1 Substances 8 2.2.2 Foods 9 2.3 Limit values (GroentenFruit Huis) 9 2.4 Additional data for CRA used in MCRA 10 2.4.1 Overview data for cumulative risk assessment 10 2.4.2 Health effects and assessment groups data 11 2.4.3 Consumption data 12 2.4.4 Processing factor data 13 2.4.5 Unit variability data 13 2.4.6 Residue definition data 13 2.4.7 Food translation data (reverse yield factors) 14 Contents Summary 5 1 Introduction 6 1.1 Overview and aim of the study 6 1.2 Development of a web portal to link private and public data 6 2 Data 8 2.1 Monitoring data (Food Compass) 8 2.2 Catalogues of substances and food products (GroentenFruit Huis) 8 2.2.1 Substances 8 2.2.2 Foods 9 2.3 Limit values (GroentenFruit Huis) 9 2.4 Additional data for CRA used in MCRA 10 2.4.1 Overview data for cumulative risk assessment 10 2.4.2 Health effects and assessment groups data 11 2.4.3 Consumption data 12 2.4.4 Processing factor data 13 2.4.5 Unit variability data 13 2.4.6 Residue definition data 13 2.4.7 Food translation data (reverse yield factors) 14 3 Method 15 3.1 Single substance-food assessments 15 3.1.1 MRL exceedance 15 3.1.2 ARfD exceedance 15 3.1.3 MRL and ARfD exceedance 15 3.2 Probabilistic cumulative risk assessments 15 3.2.1 General 16 3.2.2 Concentration modelling and occurrence frequencies 16 3.2.3 Extrapolation of food samples 16 3.2.4 Substances conversion 16 3.3 Software: the MCRA platform 17 3.4 Software: the IPGF portal 18 4 Results 22 4.1 Trend analysis for Dutch children 2013-2020 22 4.1.1 Single-substance assessments, business as usual 22 4.1.2 Cumulative risk assessments using MCRA 23 4.2 Example of detailed results (year 2015– Developmental effects) 29 5 Discussion and conclusions 34 6 References 36 Supplementary material 3 Summary Cumulative intake of mixtures of pesticide residues through consumption of fruits and vegetables can lead to health risks that are not controlled under the current EU system using only the maximum residue limit (MRL) and acute reference dose (ARfD) for single substances on single food products. In a collaboration between the European Commission, the European Food Safety Authority and member states, methods have been developed to implement Regulation (EC) No 1107/2009 that states that plant protection products should not have harmful health effects, also taking into account possible cumulative and synergistic effects. These methods have been implemented in the Monte Carlo Risk Assessment (MCRA) software which is available for national and European public institutions to perform pesticides mixture risk assessment. This report describes a web portal that was developed to allow cumulative risk assessment by the Dutch private vegetable and fruit sector organised in the Foundation Food Compass using MCRA with the monitoring data collected in the Food Compass database. A case study was performed to combine Food Compass monitoring data from the years 2013-2020 with consumption data of children from the Dutch national food consumption survey. The main interest was to assess the risk of cumulative exposure due to the combined intake of multiple pesticide residues in their diet. If potential risks were observed, it was of interest to know which residues and foods contributed to such risks and if there were trends over the period of the monitoring. The results of the cumulative assessments were compared to an analysis of limit value exceedances at the level of single measurement results. The results presented in this report are provisional due to insufficient availability of data. Some of the analytical scopes in the Food Compass database could not be linked appropriately to the active substance groups used for cumulative risk assessment. Food processing steps, such as peeling or juicing of citrus fruits, are expected to reduce residue levels, but the collection of processing factors to account for this in the calculations was incomplete. Limit values are sometimes changed, and recently artificially low limit values were introduced in the sectoral system to generate alerts for substances which have been classified as genotoxic. Such data will then also have an artificial impact on the cumulative assessments. The conventional analysis of single residue measurements showed that 1-3% of residue levels exceeded the MRL throughout the period 2013-2020. Method 3.1 Single substance-food assessments 15 3.1.1 MRL exceedance 15 3.1.2 ARfD exceedance 15 3.1.3 MRL and ARfD exceedance 15 3.2 Probabilistic cumulative risk assessments 15 3.2.1 General 16 3.2.2 Concentration modelling and occurrence frequencies 16 3.2.3 Extrapolation of food samples 16 3.2.4 Substances conversion 16 3.3 Software: the MCRA platform 17 3.4 Software: the IPGF portal 18 4 Results 22 4.1 Trend analysis for Dutch children 2013-2020 22 4.1.1 Single-substance assessments, business as usual 22 4.1.2 Cumulative risk assessments using MCRA 23 4.2 Example of detailed results (year 2015– Developmental effects) 29 5 Discussion and conclusions 34 6 References 36 Supplementary material 4 4 Summary However, the frequency of conservatively estimated exceedances of the ARfD using the PRIMo 3.1 model decreased from around 3% to below 1% in the same period. It was also found that ARfD exceedances did often occur without an associated MRL exceedance. The cumulative assessments indicated that the probability of a critical acute exposure was estimated to be in the range 0.1- 0.4% during the period 2013-2019 (the results for 2020 were affected by an artificial low ARfD value and are therefore not useful to estimate real risk). Nevertheless, the main identified risk drivers were occurrences of chlorpyrifos and chlorpyrifos-methyl in some citrus fruit products such as juices for which no account of processing effects was included in the assessment due to lack of validated data. Including validated processing factors for citrus fruit products as they are consumed in practice will result in more realistic critical probabilities which are expected to be lower. It is planned to update the current trend analysis in further work. 1.1 Overview and aim of the study A case study was performed to combine Food Compass monitoring data from the years 2013-2020 with consumption data of Dutch children. The main interest is to find if there could have been cumulative exposure due to the combined effects of multiple pesticide residues in their diet. If so, it was of interest to know which residues and foods contributed to such risks and if there were trends over the period of the monitoring. The results presented in this report are provisional due to insufficient availability of certain data. This case study is part of a project to use private sector pesticide residue monitoring data for cumulative risk assessment using public food consumption data and the publicly developed Monte Carlo Risk Assessment (MCRA) web platform. For linking private and public data the IPGF portal was developed in the context of a public-private partnership project between WUR Biometris, financed from public means, and Food Compass, who contributed the monitoring data. IPGF is the abbreviation of ‘Impactanalyse Pesticiden in Groenten en Fruit’ (‘Impact analysis Pesticides in Fruit and Vegetables’). The primary stakeholders of this work are the project partners WUR Biometris and Foundation Food Compass. WUR Biometris aims to build up expertise regarding the linkage of knowledge (seen as data) and models across the internet in an interoperable manner, and to promote the distribution of knowledge about performing cumulative risk assessments using the MCRA software. Food Compass aims to be able to perform EU compatible cumulative risk assessments on Food Compass samples per period, compare results across periods (trend analysis), to use the results for risk communication to Food Compass and GroentenFruit Huis colleagues, and possibly to Food Compass participants and/or retail representatives regarding the health impact of cumulative pesticide exposure. A secondary objective is to prepare for analyses at the sample level by Food Compass participants in the context of quality control or early warning systems. The longer-term aim is to create better links between private and public knowledge management systems in the interest of open and transparent risk assessment. 5 5 2.1 Monitoring data (Food Compass) Food Compass monitoring data for the years 2012-2020 were obtained from the Food Compass Web API in December 2021. The samples used in the case study were gathered from all Food Compass sampling programs: "Monitoring (M)", "EWRS (X)", "NVWA (N)", "Bedrijfseigen monster (E)" and "Aanvullend monster (A)". Food Compass food sample reports were retrieved for each batch and aligned with the known laboratory scopes. The latter is required because the sample reports contain only information on the positive substance concentrations and not the substance measurements that were below the detection limit. The Laboratory scope lists provide information on all substances measured by a given analytical method, including the applicable detection limits. The import step also contains some validation and curation steps to detect and, if possible, restore inconsistencies between the known laboratory scopes and the reported sample substance concentrations. Inconsistencies are reported as critical or non-critical measurement inconsistencies (reflecting individual substance measurements within a sample) and inconsistencies in linking the reported analysis methods with the known analytical scopes. For the cumulative exposure assessments, the imported samples are converted to the MCRA concentration data format. The table below shows the results of the sample imports forming the concentration data for the batch exposure assessments. Table 1 Food samples of the batches imported from Food Compass. Table 1 Food samples of the batches imported from Food Compass. Year Total samples Number of sampled food products Samples1 with invalid analytical scopes Samples1 with invalid measurements Samples with non-critical measurement inconsistencies Samples available for cumulative analyses Samples with positive concentrations for cumulative analyses 2013 4376 171 259 470 390 4329 2622 2014 3771 158 424 396 394 3626 2389 2015 3732 166 134 514 408 3650 2270 2016 3244 160 105 351 420 3226 2129 2017 3372 160 640 446 329 3366 2014 2018 2165 147 354 343 267 2120 1321 2019 1710 156 460 286 228 1564 1051 2020 1472 143 263 238 182 1387 959 1 These samples may still contain valid data for other analysis methods/substances. Residue monitoring data were listed with 3 laboratories. The complete list of 37 laboratories had in total 675 different laboratory scopes. Residue monitoring data were listed with 3 laboratories. The complete list of 37 laboratories had in total 675 different laboratory scopes. 2.2 Catalogues of substances and food products (GroentenFruit Huis) 2.2.1 Substances 1.2 Development of a web portal to link private and public data The IPGF web portal is developed to analyse the concentration levels of substance residues found on the fresh fruit and vegetable samples recorded by Food Compass and assess the human health risk associated with consumption of fruits and vegetables with such residue concentration levels. Users of the portal are able to evaluate the substance residue concentration levels of Food Compass samples and 1) compare these levels with legal residue limits (MRLs), 2) compare these levels with non- statutory retail requirements, and 3) evaluate the potential human health risk from exposure to these concentration levels using different assessment models. The latter comprises both the single-substance, deterministic IESTI calculations, and more realistic multi-substance, probabilistic cumulative exposure and risk calculations as available in MCRA. In addition, users are able to evaluate the trends of the substance residue concentration levels and their potential associated impact on human health over time. The IPGF portal retrieves the Food Compass concentration data from the Food Compass database, which can be done via the Food Compass web API. These 6 concentration data are validated, curated, and linked with other data as a prerequisite for performing the analyses. E.g., linking of food codes and curation of analytical scopes. Therefore, the portal includes a data management module to allow for data inspection and, if needed, data curation of some identified data types. concentration data are validated, curated, and linked with other data as a prerequisite for performing the analyses. E.g., linking of food codes and curation of analytical scopes. Therefore, the portal includes a data management module to allow for data inspection and, if needed, data curation of some identified data types. The potential users of the IPGF portal are Food Compass and GroentenFruit Huis staff members, Food Compass participants (for single sample analyses), and interested stakeholders in the Netherlands or in Europe. To allow usage by a broad audience, the portal has been developed in English. 7 Residue monitoring data were listed with 3 laboratories. The complete list of 37 laboratories had in total 675 different laboratory scopes. These samples may still contain valid data for other analysis methods/substances. 2.2.2 Foods The food catalogue was downloaded from the GroentenFruit Huis WebAPI on 01-12- 2021. The food catalogue was adapted and stored in the IPGF portal by mapping to EFSA MATRIX codes which were used as modelled foods for cumulative assessments. The final food catalogue has 495 entries. 2.2.1 Substances The substance catalogue was downloaded from the GroentenFruit Huis WebAPI on 01- 12-2021. Substances are identified by GroentenFruit Huis codes, which are mostly the same as CAS codes but are adapted in some cases. The substance catalogue was 8 adapted and stored in the IPGF portal by mapping the GroentenFruit Huis codes to CAS codes and then to EFSA PARAM codes. The final substance catalogue has 1411 entries. adapted and stored in the IPGF portal by mapping the GroentenFruit Huis codes to CAS codes and then to EFSA PARAM codes. The final substance catalogue has 1411 entries. 1 0.0001 mg/kg bw/day is an artificial low value meant to generate ARfD exceedance signals if this substance is found. The substance has been declared to be genotoxic mutagenic and therefore is no longer allowed according to the interpretation by NVWA. g p y 2.3 Limit values (GroentenFruit Huis) In those cases the earliest available ARfD value was used. 2.3 Limit values (GroentenFruit Huis) A database with MRL and ARfD values for substance/food combinations is maintained at the GroentenFruit Huis portal and is periodically synchronised with the EU Pesticide database. For the sample calculations, the GroentenFruit Huis web service is used to get the MRL for positive substance concentrations of the samples. Acute Reference Dose (ARfD) values used in this study both for single sample calculations and batch calculations were a combination of values obtained from the GroentenFruit Huis web service on 01-12-2021 (the most recent values) and values that were obtained from earlier versions of the GroentenFruit Huis acceptance environment. All ARfD values were labelled with a ‘Valid from’ date. Some examples are shown in Table 2. In the regulatory system, ARfD values are only set for active substances that are supposed to have no health effects below a certain threshold. ARfD values are not derived for active substances with known or presumed health effects due to a non- threshold mode of action, such as substances that are carcinogenic, mutagenic, or toxic for reproduction (CMR substances). In practice, this means that such active substances are or will be excluded from the market. However, some of these substances were allowed in previous years and are therefore found in historical monitoring data. To obtain a clear alert for current use, GroentenFruit Huis has decided to include an artificial very low value for such substances. Relevant for the current case study, an artificial low ARfD of 0.0001 mg/kg bw/day was set for Chlorpyrifos and Chlorpyrifos-methyl per 13-11-2020. Table 2. Examples Acute Reference Doses for selected substances. Substance code Substance name ARfD (mg/kg bw/day) valid from 133062 captan 0.3 11-07-2008 101213 chlorprofam 0.5 02-03-2004 2921882 chlorpyrifos 0.005 07-04-20142 2921882 chlorpyrifos 0.00011 13-11-2020 5598130 chlorpyrifos-methyl 0.1 21-10-2005 5598130 chlorpyrifos-methyl 0.00011 13-11-2020 16672870 ethephon 0.05 02-12-2008 35554440 imazalil 0.05 04-03-2010 91465086 lambda-cyhalothrin 0.005 01-04-20162 2032657 methiocarb 0.013 07-02-2007 60207901 propiconazole 0.3 17-07-2003 60207901 propiconazole 0.1 19-06-2019 175013180 pyraclostrobin 0.03 10-03-2004 107534963 tebuconazole 0.03 25-09-2008 1 0.0001 mg/kg bw/day is an artificial low value meant to generate ARfD exceedance signals if this substance is found. The substance has been declared to be genotoxic mutagenic and therefore is no longer allowed according to the interpretation by NVWA. Table 2. Examples Acute Reference Doses for selected substances. 9 2 Is some cases the earliest Valid from date available was later than the starting date of the trend analysis, i.e. 01-01-2013. 2.4 Additional data for CRA used in MCRA 2.4.1 Overview data for cumulative risk assessment 2.4.1 Overview data for cumulative risk assessment The data required for cumulative risk assessments in MCRA originate from different sources. The IPGF portal feeds parts of the data to MCRA, for example the concentration data, and specifies the data to be used for calculation jobs. Some data are already available at MCRA and can be used in assessments by just referencing these datasets, for example the consumption data used for the assessments. The table below summarizes that data needs for performing the cumulative risk assessments. Table 3 Data for cumulative risk assessment in MCRA. Data type Data Coding systems Data origin Recoding needed Foods (and processing types) All food products measured in the concentration data and all food products with consumptions in the Raw Primary Commodity consumption data. EFSA MATRIX food product codes with processed foods coded with FoodEx2 facet codes for the processing types. MCRA No Substances All FC substances with additional PARAM codes from CAG definitions and residue definitions Substance codes following the FC CAS coding system. IPGF portal / original data at GroentenFruit Huis No Effects Acute organ level CAGs, derived from Nielsen et al. (2012). Custom effect coding system used in the CAGs dataset. MCRA No Active substances (assessment group memberships) Acute organ level CAGs, derived from Nielsen et al. (2012). Substance codes following the FC CAS coding system and the custom effect coding system used in the CAGs dataset. MCRA EFSA PARAM to FC Cas Consumptions RPC consumption data of: • NL VCP child population 2005- 2006 (2-6yr) • NL VCP general population (age 7- 69) 2007-2010 • NL VCP elderly population (70+ yr) 2010-2012 EFSA MATRIX food product codes with processed foods coded with FoodEx2 facet codes for the processing types. MCRA No Concentrations (background) FC concentration data (specific sample selection unknown) EFSA MATRIX food product codes obtained by mapping of the FC food codes. Substance codes following the FC CAS coding system. IPGF portal / original data at FC FC food codes to EFSA MATRIX codes Residue definitions Residue definitions from EFSA/RIVM-FPA Substance codes following the FC CAS coding system. IPGF portal EFSA PARAM to FC Cas Table 3 Data for cumulative risk assessment in MCRA. 10 Processing factors Import of RIVM processing factors EFSA MATRIX food product codes. FoodEx2 facet codes for the processing types. Substance codes following the FC CAS coding system. 2.4.1 Overview data for cumulative risk assessment IPGF portal / original data at MCRA EFSA PARAM to FC Cas Unit variability factors EFSA/RIVM-FPA Tier II unit variability factors EFSA MATRIX food product codes. FoodEx2 facet codes for the processing types. MCRA No Food translations Food translations containing the RPC yield factors for processed foods EFSA MATRIX food product codes. FoodEx2 facet codes for the processing types. MCRA No Hazard characterisations ARfDs from GroentenFruit Huis Substance codes following the FC CAS coding system. IPGF portal / original data at GroentenFruit Huis No 2.4.2 Health effects and assessment groups data 2.4.2 Health effects and assessment groups data Cumulative exposure assessments were performed for 15 adverse effects at organ level, with cumulative assessment groups (CAGs) of varying sizes. These CAGs were proposed by Nielsen et al. (2012) in a scientific opinion for the EFSA Panel on Plant Protection Products and their Residues (PPR). It should be noted, that EFSA has started a process for more data collection and an updated definition of CAGs, but this has until now resulted in just three CAGs for acute effects (EFSA 2019c, 2020). For illustrative purposes the 15 CAGs from Nielsen et al. (2012) were selected in the context of this case study. An MCRA effects and assessment groups dataset is created from the CAGs proposed by Nielsen et al. (2012). The acute Effects and CAGs at CAG level 1 (organ level) will be used for the analyses. I.e., an analysis will be done for each level 1 effect/CAG. The CAG dataset is available on a share in MCRA and a local copy of this data is maintained within the portal for administration/quality checking. Table 4 Health effects and assessment groups for cumulative risk assessment. Effect Description Substances in CAG Substances with missing ARfD Index substance Adrenal Adverse effects on the adrenal gland 10 fosthiazate (98886443) Bone Adverse effects on the bone marrow 9 bromoxynil (1689845) Cardiovascular Adverse effects on the cardiovascular system 10 formetanate (22259309) Developmental Adverse developmental effects 110 fenoxaprop-P-ethyl oxamyl (23135220) Eye Adverse effects on the eye 39 oxamyl (23135220) Haematological Adverse effects on the haematological system 68 fenoxaprop-P-ethyl methomyl (16752775) Kidney Adverse effects on the kidney 47 fenoxaprop-P-ethyl oxamyl (23135220) Liver Adverse effects on the liver 100 fenoxaprop-P-ethyl dinocap (39300453) Muscle Adverse effects on the muscle 10 fosthiazate (98886443) Nervous Adverse effects on the nervous system 54 oxamyl (23135220) Table 4 Health effects and assessment groups for cumulative risk assessment. 2.4.1 Overview data for cumulative risk assessment 11 Parathyroid Adverse effects on the parathyroid 5 methconazole (125116236) Skeleton Adverse effects on the skeleton 3 tetraconazole (112281773) Spleen Adverse effects on the spleen 9 tetraconazole (112281773) Thyroid Adverse effects on the thyroid 32 dinocap (39300453) Urinary Adverse urinary effects 14 flusilazole (85509199) 2.4.3 Consumption data Assessments use the consumption data from three Dutch food surveys (VCP) for three different subpopulation the child population (2-6yr) 2005-2006, the general population (age 7-69) 2007-2010, and the elderly population (70+yr) 2010-2012. In the current report only the consumption data for children have been used to have a first demonstration of the results. The data has been provided by RIVM to EFSA and has been provided again by EFSA in the form of raw primary commodity consumption data (RPC, EFSA 2019a), meaning that the consumptions are expressed in terms of the raw (measured) food products. A number of modelled foods found in the foods catalogue did not match/align with the RPF consumption data and were therefore not included in the cumulative assessments (Table 5). Table 5 Modelled/measured foods not matched with the consumption data. Modelled food (MATRIX) code Modelled food name P0252030A Chards/beet leaves P0163050A Granate apples/pomegranates P0161060A Kaki/Japanese persimmons P0110040A Limes P0231040A Okra (lady's fingers) P0161050A Carambolas P0162040A Prickly pears P0213050A Jerusalem artichokes P0213060A Parsnips P0213090A Salsifies P0213110A Turnips P0232990A Other cucurbits with edible peel P0233990A Other cucurbits with inedible peel P0255000A Witloofs/Belgian endives P0840020A Ginger P0163040A Papayas P0260030A Peas (with pods) P0252020A Purslanes P0130030A Quinces P0270070A Rhubarbs P0251060A Roman rocket/rucola Parathyroid Adverse effects on the parathyroid 5 methconazole (125116236) Skeleton Adverse effects on the skeleton 3 tetraconazole (112281773) Spleen Adverse effects on the spleen 9 tetraconazole (112281773) Thyroid Adverse effects on the thyroid 32 dinocap (39300453) Urinary Adverse urinary effects 14 flusilazole (85509199) 2.4.3 Consumption data Assessments use the consumption data from three Dutch food surveys (VCP) for three different subpopulation the child population (2-6yr) 2005-2006, the general population (age 7-69) 2007-2010, and the elderly population (70+yr) 2010-2012. In the current report only the consumption data for children have been used to have a first demonstration of the results. The data has been provided by RIVM to EFSA and has been provided again by EFSA in the form of raw primary commodity consumption data (RPC, EFSA 2019a), meaning that the consumptions are expressed in terms of the raw (measured) food products. A number of modelled foods found in the foods catalogue did not match/align with the RPF consumption data and were therefore not included in the cumulative assessments (Table 5). Table 5 Modelled/measured foods not matched with the consumption data. Modelled food (MATRIX) code Modelled food name P0252030A Chards/beet leaves P0163050A Granate apples/pomegranates P0161060A Kaki/Japanese persimmons P0110040A Limes P0231040A Okra (lady's fingers) P0161050A Carambolas P0162040A Prickly pears P0213050A Jerusalem artichokes P0213060A Parsnips P0213090A Salsifies P0213110A Turnips P0232990A Other cucurbits with edible peel P0233990A Other cucurbits with inedible peel P0255000A Witloofs/Belgian endives P0840020A Ginger P0163040A Papayas P0260030A Peas (with pods) P0252020A Purslanes P0130030A Quinces P0270070A Rhubarbs P0251060A Roman rocket/rucola P0212020A Sweet potatoes P0256100A Tarragon The RPC consumption datasets are available for use on a share on MCRA and a reference to these dataset is sufficient for using it is an MCRA cumulative exposure analysis from the IPGF portal. Table 5 Modelled/measured foods not matched with the consumption data. The RPC consumption datasets are available for use on a share on MCRA and a reference to these dataset is sufficient for using it is an MCRA cumulative exposure analysis from the IPGF portal. 12 2.4.4 2.4.4 Processing factor data The EFSA database of processing factors prepared by Scholtz et al. (2018) serves as the basis of the processing factors used in this case study. However, this dataset contains only a limited amount of substance-food combinations. In this project, we noticed in initial assessments that imazalil on citrus fruits was identified as an important risk driver, but that peeling of citrus fruits is expected to remove most of the imazalil residues. 2.4.3 Consumption data More processing factors are available in a Dutch database maintained at RIVM in the last updated version of 20201, but the latter database is not organised using the harmonised substance and food codes at EFSA and could therefore not be used automatically. For the analyses reported here, the EFSA processing factor data were extended with processing factors for imazalil in citrus food as were available from the RIVM database. For preparing the dataset, the EFSA PARAM codes used by the original processing factors dataset of Scholtz et al. (2018) were mapped to the Food Compass substance coding system (based on CAS) using the mapping as available in the internal substances catalogue. A data share in MCRA contains this generated dataset processing factor dataset, which can be referenced for use in cumulative exposure assessments in MCRA. 2.4.5 2.4.5 Unit variability data The same unit variability are used as used in the Tier II calculations in van Klaveren et al. (2019a) and EFSA (2020a). It should be noted that these studies focused on a subset of 30 food products and no unit variability factors are available for the food products not considered by these studies. The unit variability factors dataset is available for use on a share on MCRA and a reference to this dataset is used within the cumulative exposure analyses in MCRA. 2.4.6 The cumulative exposure assessments are performed at the level of so-called active substances, which are the substances that are associated with the effects and CAGs and for which potency information is assumed to be available. Substance conversions are used for converting measured substance concentrations (such as sum-substance measurements) to active substance concentrations. The substance conversions are obtained from the Food Compass substances hierarchy, which is included in the substances catalogue. For each sum-substance that is linked to one or more active substances, substance conversion rules are added to map concentration values of the sum-substances to active substance concentrations. These conversion rules specif y the proportion of measurements of the sum-substance measurements that can be assumed to translate exclusively to a concentration of each active substance, and a conversion factor to translate the concentration of the sum-substance to a concentration of the active substance. Due to a lack of data, a conversion factor of 1 is assumed for all rules and equal proportions of 1/n are assumed for all active substances linking to the sum- substance, with n being the total number of substances linking to the sum-substance. As an example, consider the dithiocarbamates substances in the table below. The sum- substance (dithiocarbamaten (som als CS2)) links to four active substances. For these active substances, four substance conversion rules are created. Each with a proportion of 0.25 and a conversion factor of 1 (see table below). Table 6 Example: the substance hierarchy of the dithiocarbamates in the substances catalogue. Substance code Substance name Type EFSA PARAM code Is sum (Y/N) Sum substance code 1 https://www.rivm.nl/en/chemkap/fruit-and-vegetables/processing-factors, last update 11 June 2020 le 6 Example: the substance hierarchy of the dithiocarbamates in the substances catalogue. 13 75150 carbondisulfide (CS2) BreakdownPro duct RF-0151-005- PPP N 90000075150 8018017 mancozeb BreakdownPro duct RF-0151-004- PPP N 90000075150 9006422 metiram BreakdownPro duct RF-0151-002- PPP N 90000075150 12427382 maneb BreakdownPro duct RF-0151-003- PPP N 90000075150 900000751 50 dithiocarbamaten (sum as CS2) Residue RF-0151-001- PPP Y Table 7 Example: substance conversion rules for dithiocarbamates as generated from the substances hierarchy. 3.1.3 MRL and ARfD exceedance 3.1.3 MRL and ARfD exceedance MRL and ARfD exceedance In practice, Food Compass is using MRL exceedance as a first screening and ARfD exceedances are registered for those samples where MRL was exceeded. 3 Method 3 3.1.1 MRL exceedance 3.1.1 MRL exceedance Residue levels for single substances in crop samples are compared to the MRL for that substance in that crop and exceedances are reported. The %MRL calculation is delegated to the GroentenFruit Huis web service. For each positive substance concentration of each sample, the GroentenFruit Huis web service is used to compare the measured concentration to the MRL as stored in the GroenFruit Huis portal in the form of a percentage (%MRL). 3.1.2 ARfD exceedance For GroentenFruit Huis members, available at https://groentenfruithuis.nl/dashboard under Tools - Voedselveiligheid. 3.1 Single substance-food assessments In the business-as-usual scenario, risk assessment by Food Compass focuses on the inspection of individual concentration values in the Food Compass monitoring data. 2.4.6 idMeasuredSubstance idActiveSubstance ConversionFactor IsExclusive Proportion 90000075150 75150 1 TRUE 0.25 90000075150 9006422 1 TRUE 0.25 90000075150 8018017 1 TRUE 0.25 90000075150 12427382 1 TRUE 0.25 For each cumulative exposure assessment, a substance conversions dataset is created in this way uploaded to MCRA and used in the assessment Table 7 Example: substance conversion rules for dithiocarbamates as generated from the substances hierarchy. For each cumulative exposure assessment, a substance conversions dataset is created in this way, uploaded to MCRA, and used in the assessment. For each cumulative exposure assessment, a substance conversions dataset is created in this way, uploaded to MCRA, and used in the assessment. 2.4.7 Food translation data (reverse yield factors) 2.4.7 Food translation data (reverse yield factors) The food consumptions of the food survey are specified at the level of processed raw commodities. Within the cumulative exposure assessments, these consumptions are linked to the measured (raw) food products using food translations. The food translations do not only qualitatively link the processed foods to the unprocessed/raw foods, but also include weight correction factors to translate consumed food amounts to equivalent modelled food amounts. For the RPC consumption data, the translations data consists of the weight correction factors due to processing. The food translation dataset is available for use on a share on MCRA and a reference to this dataset is sufficient for using it is an MCRA cumulative exposure analysis from the IPGF portal. 14 3.1.2 ARfD exceedance The exposure of the substance via consumption of the crop can be estimated using the IESTI model according to the PRIMo 3.1 specification (EFSA 2019b) and compared to the ARfD as stored in the GroenFruit Huis portal using the ARfD% application of GroentenFruit Huis2. The %ARfD calculation is delegated to the GroentenFruit Huis web service, which computes the %ARfD with the consumption amounts and nominal bodyweights of the critical population and the currently active ARfD value. For the retrospective analyses in the IPGF portal, it is also desirable to compute the %ARfD for historical samples, using the then-present ARfD value. Therefore, the %ARfD value received from GroentenFruit Huis is recomputed for historical samples by dividing by the currently active ARfD and multiplying with the ARfD active during the period of sampling. In the GroentenFruit Huis tool, artificial low ARfD values were included for some genotoxic substances such as chlorpyrifos and chlorpyridos-methyl (see section 2.3). Moreover, for these cases all processing factors were removed from the calculation in the tool. 2 For GroentenFruit Huis members, available at https://groentenfruithuis.nl/dashboard under Tools - Voedselveiligheid. 3.2.2 3.2.2 Concentration modelling and occurrence frequencies Concentration modelling is done according to the EC 2018 Tier 2 specifications. Thus, sample-based concentration modelling is done, non-detects are replaced by 1/2 x LOR and missing values are imputed using occurrence frequency estimates. Occurrence patterns and frequencies are computed the same way as in the EC 2018 Tier 2 method, except that no substance authorisation data to restrict use percentage up-scaling to authorised uses was used. This is because authorised uses data was not sufficiently available to use this option. 3.2 Probabilistic cumulative risk assessments Cumulative effects from mixtures of pesticide residues can lead to health risks that are not controlled under the current EU system using only the MRL and ARfD for single substances. In a collaboration between the European Commission, the European Food Safety Authority and member states methods have been developed to assess cumulative exposure and risk (van Klaveren et al. 2019ab; EFSA 2020ab). These methods have been implemented in MCRA (van Klaveren et al. 2019ab). In the current study we consider risk for acute health effects as might result from consuming fruit and vegetables from the Dutch market against a background of other dietary consumptions of Dutch children. Specifically, we apply the EC Tier 2 method as was proposed by the European Commission in 2018 and was subsequently adopted by 15 EFSA. For a full description of the probabilistic method for acute health effects see van Klaveren et al. (2019a). Here we only provide a short summary. EFSA. For a full description of the probabilistic method for acute health effects see van Klaveren et al. (2019a). Here we only provide a short summary. Health effects can be grouped according to hierarchical levels. RIVM and EFSA have thus far applied cumulative risk assessments for four specific phenomenological effects, two neurological effects and two effects on the thyroid (level 2; van Klaveren et al. 2019ab, EFSA 2020ab), but have also investigated cumulative risk assessments at the corresponding organ levels, neurological and thyroid (level 1; te Biesebeek et al., 2021). In this case study, cumulative modelling is applied for 15 cumulative assessment groups (CAGs) defined at level 1 by Nielsen et al. (2012). 3.2.3 No extrapolation was done of food samples for foods with a limited amount of samples (data poor foods) from other foods (data rich foods). The reason for this is that the extrapolation rules were not sufficiently available. 3.2.1 The exposure and risk assessment are computed in an acute MCRA risk assessment using the hazard index as the risk metric. Dietary exposures are computed in principle according to EC 2018 Tier 2 settings; meaning that simulated substance residues are generated using a sample-based approach, processing factors are used in the calculation, and unit-variability is accounted for in a beta-binomial model using a realistic estimates nature. The active substances of the assessment are obtained from data, with a further restriction to only the substances for which an ARfD is available. The hazard characterisations are formed by ARfDs obtained from GroentenFruit Huis, which apply to the critical effect that are used as a proxy for specific (organ / CAG level 1) effects in the cumulative exposure assessments. This is similar to the approach followed by te Biesebeek et al. (2021). For each exposure assessment, the most toxic substance (i.e., the substance with the lowest ARfD) was selected as reference substance. Options are available to run the cumulative exposure and risk assessments with or without uncertainty. More specifically, there are two options for uncertainty analysis: an uncertainty-test option using only 10 uncertainty analysis cycles (bootstrap cycles), using a reduced population size of 10.000 simulated individuals in the uncertainty cycles, and an uncertainty-full option with 100 bootstrap cycles simulating 100.000 individuals in each bootstrap run. The active substances of the assessment are obtained from data, with a further restriction to only the substances for which an ARfD is available. The hazard characterisations are formed by ARfDs obtained from GroentenFruit Huis, which apply to the critical effect that are used as a proxy for specific (organ / CAG level 1) effects in the cumulative exposure assessments. This is similar to the approach followed by te Biesebeek et al. (2021). For each exposure assessment, the most toxic substance (i.e., the substance with the lowest ARfD) was selected as reference substance. Options are available to run the cumulative exposure and risk assessments with or without uncertainty. More specifically, there are two options for uncertainty analysis: an uncertainty-test option using only 10 uncertainty analysis cycles (bootstrap cycles), using a reduced population size of 10.000 simulated individuals in the uncertainty cycles, and an uncertainty-full option with 100 bootstrap cycles simulating 100.000 individuals in each bootstrap run. 3.2.4 Substances conversion Substances conversion rules are used to translate measured substance concentrations (e.g., of sum substances) to active substance concentrations. The residue definitions are obtained from the sum-substance hierarchy information of the Food Compass substances catalogue. However, substance authorisation information is not included in the substance conversion, since this information was not sufficiently available. Note that samples can have multiple sample analyses, measured using different analytical methods. Because of this, it may be possible that there are multiple (conflicting) substance measurements for the same substance if the substance is by both analytical methods. This may also occur indirectly (via active substance 16 allocation) when one analytical method reports the active substance concentration directly and another analytical method reports a sum-substance concentration that translates that active substance. allocation) when one analytical method reports the active substance concentration directly and another analytical method reports a sum-substance concentration that translates that active substance. As an example, consider the following example: As an example, consider the following example: • A sample can be analysed with both LC-MS and GC-MS. • The LC-MS method measures bromoxynil(sum), translating to bromoxynil (as) and bromoxynil-octanoate. • The LC-MS method measures bromoxynil(sum), translating to bromoxynil (as) and bromoxynil-octanoate. • The GC-MS method measures bromoxynil (as) and bromoxynil-octanoate directly • The GC-MS method measures bromoxynil (as) and bromoxynil-octanoate directly. • Hence, active substance allocation leads to two (possibly conflicting) concentration values for the active substances bromoxynil (as) and bromoxynil-octanoate on the same sample. The GC MS method measures bromoxynil (as) and bromoxynil octanoate directly. • Hence, active substance allocation leads to two (possibly conflicting) concentration values for the active substances bromoxynil (as) and bromoxynil-octanoate on the same sample. If active substance allocation leads to multiple allocated measurements for the same substance on the same sample, then the following procedure is implemented rules for resolving these inconsistencies: • If all measurements are non-detect, then select the measurement with the smallest LOR. • If any of the measurements is positive or zero, then take the mean of all positive/zero measurements. Note that these rules are quite generic and would work quite well also in case there are many measurements for the same active substance. In practice, one would expect only a few (two). 3 The offical version of MCRA 9.1 can be found at https://mcra.rivm.nl. In the current report calculations were made using the test verison of MCRA 9.1 at WUR, which is in principle the same version as available at RIVM. 3.3 Software: the MCRA platform The cumulative risk calculations were performed using the Monte Carlo Risk Assessment (MCRA) portal, version 9.13. MCRA 9, also known as the EuroMix Toolbox, is a program for Monte Carlo Risk Assessment, developed for RIVM by Wageningen University & Research, Biometris to facilitate RIVM's tasks for the Dutch food safety authority (NVWA) and for cooperation in international projects (EFSA, EC Research). MCRA 9 was developed in the EuroMix project and in collaborations with EFSA. For acute dietary risk assessment of pesticides, MCRA provides functionality to link consumption data from a dietary survey and residue occurrence data. Consumption of individual-days are randomly combined with residue levels for all consumed foods to produce an estimate of the exposure distribution. Scaled against the ARfD of a substance the exposure distribution can be expressed as a distribution of the hazard quotient (in MCRA termed hazard index, HI), with values above 1 indicating potential risk. For cumulative assessments all residue levels of the substances in an assessment group are scaled by their relative potency factor (RPF) with respect to a selected index substance. Scaled exposures are summed, and the sum is scaled to HI by dividing by the ARfD of the index substance. The consumption data and the files defining health effects and cumulative assessment groups are available in MCRA for the Food Compass user. Many refinements of the assessment are possible, and some are part of the EC Tier 2 method in this case study. This requires additional data on processing factors, unit variability factors and residue definitions. These data are also available in MCRA to the Food Compass user. The use of MCRA requires a high level of understanding and some degree of experience. To allow the use of MCRA functionality from other more easily accessible entry points (such as the IPGF platform, see next section), an application programming interface (API) was created to allow MCRA calculations to be delivered as a web service (WebAPI). For example, external programs can ask MCRA which data is available, send 17 specific data to MCRA, ask MCRA to perform a specific calculation and to send back the results. specific data to MCRA, ask MCRA to perform a specific calculation and to send back the results. 3.4 Software: the IPGF portal All analyses of this case study were performed using a beta version of the IPGF web portal (version 2.0.0-beta.1). As mentioned, IPGF portal is a web platform specifically to designed to perform human health risk analyses on the Food Compass concentration data. It does so by linking this concentration to other data and delegating model calculations to specific modelling services, such as MCRA for cumulative risk assessment and the GroentenFruit Huis web service for IESTI and MRL calculations. The e-infrastructure of the IPGF portal is depicted in the figure below. The platform can be used to collect data from and delegate calculations to different external web services via Web APIs. These web services are the Food Compass web service, the GroentenFruit Huis web service, and the MCRA web service. Each service is used for different purposes, which is illustrated in the platform service infrastructure diagram of Figure 2. Establishing connections with these services and linking the data from multiple sources is therefore a key aspect of this portal. Figure 1 Illustration of the e-infrastructure of the communication and data exchange between the IPGF Portal and other web services. Figure 1 Illustration of the e-infrastructure of the communication and data exchange between the IPGF Portal and other web services. The portal presents three types of analyses to analyse the food samples from Food Compass: The portal presents three types of analyses to analyse the food samples from Food Compass: • Single sample analyses: analysis of individual samples, either entered manually or selected from all available samples in the FC data (findable by sample report number). This type of analyses can be performed by all users of the portal. • Sample collection (batch) analyses: analysis of a collections of samples. Consider all samples in a specified time period (e.g. the year 2019). Batches can be defined (by means of a start date, end date, etc.) and run by administrators of the portal. All users are able to evaluate the results of batch analyses. y • Batch comparison analyses: comparison of (risk) indicators of different batch analyses. Particularly intended to visualize trends in time. 18 For the present case study, the batch analysis and the batch comparison analysis are used. Figure 2 shows a screenshot of the batch overview page. 3.4 Software: the IPGF portal On this page, main information about the batch and the analysis status is shown and from this page the user can browse to the various batch result report pages. Figure 3 shows a screenshot of the cumulative exposure assessment results of this batch. For each health effect, it shows the cumulative exposure and confidence intervals at a specified percentile, and the probability of critical exposure (POCE), with its confidence intervals. As a last example, Figure 4 shows the main results of multiple batches combined in an overview table, which is part of the batch comparison analyses. In the case study of this report, a batch analysis was done for each year from 2013 to 2020 and the results were extracted from the batch analysis reports and the batch comparison report. In the case study of this report, a batch analysis was done for each year from 2013 to 2020 and the results were extracted from the batch analysis reports and the batch comparison report. In the case study of this report, a batch analysis was done for each year from 2013 to 2020 and the results were extracted from the batch analysis reports and the batch comparison report. Figure 2 Screenshot of the IPGF web portal. This is the batch analysis overview page of the batch "2014 - Children". comparison report. Figure 2 Screenshot of the IPGF web portal. This is the batch analysis overview page of the batch "2014 - Children". Figure 2 Screenshot of the IPGF web portal. This is the batch analysis overview page of the ba "2014 - Children". Figure 2 Screenshot of the IPGF web portal. This is the batch analysis overview page of the batch "2014 - Children". 19 Figure 3 Screenshot of the provisional cumulative exposure results report of a batch analysis in the IPGF portal. Figure 3 Screenshot of the provisional cumulative exposure results report of a batch analysis in the IPGF portal. Figure 3 Screenshot of the provisional cumulative exposure results report of a batch analysis i the IPGF portal. Figure 3 Screenshot of the provisional cumulative exposure results report of a batch analysis in the IPGF portal. 20 Figure 4 Screenshot of the batch comparison report, combining the results of multiple batches in an overview table. Figure 4 Screenshot of the batch comparison report, combining the results of multiple batches in an overview table. Single-substance assessments, business as usual The percentages of samples with exceedance of the MRL or with a calculated IESTI exceeding the ARfD is shown for the years 2013-2020 in Table 8 and Table 9 and in Figure 5. Large numbers, i.e. 31-41 different foods and 45-72 different substances were involved in the MRL exceedances. Smaller but still quite large numbers, i.e. 5-22 different foods and 3-22 different substances were involved in the ARfD exceedances. The conventional analysis of single residue measurements showed that 1-3% of residue levels exceeded the MRL throughout the period 2013-2020, without a clear trend. The most notable change was the gradual decrease of the ARfD exceedance frequency using the PRIMo 3.1 model from around 3% in earlier years to 0.7 % in the latest year. Another interesting observation is the low frequency of samples with both MRL and ARfD exceedances (always below 0.5%), meaning that ARfD exceedances may also occur without an accompanying MRL exceedance in the same sample. Table 8. Single substance assessments: Concentration exceeding MRL. Year Number of samples % of samples with concentration >MRL for any substance Number of foods with at least one MRL exceedance Number of substances with at least one MRL exceedance Number of food/ substances combinations with at least one MRL exceedance 2013 4376 1.6 37 60 84 2014 3771 3.0 39 67 94 2015 3732 1.3 34 57 76 2016 3244 3.0 39 72 116 2017 3372 2.2 41 57 82 2018 2165 2.6 37 58 71 2019 1710 3.3 39 55 76 2020 1472 3.0 31 45 61 Table 9. Single substance assessments: IESTI exceeding ARfD. Year Number of samples % of samples with IESTI > ARfD for any substance Number of foods with at least one ARfD exceedance Number of substances with at least one ARfD exceedance Number of food/substances combinations with at least one ARfD exceedance % of samples with concentration > MRL and IESTI > ARfD for any substance 2013 4376 2.9 16 11 31 0.2 2014 3771 2.6 20 22 40 0.4 2015 3732 3.2 22 14 31 0.4 2016 3244 2.5 15 14 28 0.3 2017 3372 1.9 13 10 20 0.1 2018 2165 2.2 9 10 13 0.4 2019 1710 0.9 5 3 5 0.3 2020 1472 0.7 9 9 13 0.5 Table 8. Single substance assessments: Concentration exceeding MRL. Results 4 4.1.1 1 Single-substance assessments, business as usual 3.4 Software: the IPGF portal Figure 4 Screenshot of the batch comparison report, combining the results of multiple batches in an overview table. 21 Single-substance assessments, business as usual Year Number of samples % of samples with concentration >MRL for any substance Number of foods with at least one MRL exceedance Number of substances with at least one MRL exceedance Number of food/ substances combinations with at least one MRL exceedance 2013 4376 1.6 37 60 84 2014 3771 3.0 39 67 94 2015 3732 1.3 34 57 76 2016 3244 3.0 39 72 116 2017 3372 2.2 41 57 82 2018 2165 2.6 37 58 71 2019 1710 3.3 39 55 76 2020 1472 3.0 31 45 61 Table 8. Single substance assessments: Concentration exceeding MRL. Table 9. Single substance assessments: IESTI exceeding ARfD. Year Number of samples % of samples with IESTI > ARfD for any substance Number of foods with at least one ARfD exceedance Number of substances with at least one ARfD exceedance Number of food/substances combinations with at least one ARfD exceedance % of samples with concentration > MRL and IESTI > ARfD for any substance 2013 4376 2.9 16 11 31 0.2 2014 3771 2.6 20 22 40 0.4 2015 3732 3.2 22 14 31 0.4 2016 3244 2.5 15 14 28 0.3 2017 3372 1.9 13 10 20 0.1 2018 2165 2.2 9 10 13 0.4 2019 1710 0.9 5 3 5 0.3 2020 1472 0.7 9 9 13 0.5 Table 9. Single substance assessments: IESTI exceeding ARfD. 22 Figure 5. Time trends in frequency (sample percentage) of MRL and ARfD exceedances. ARfD exceedance based on single-substance point estimate calculations (IESTI in PRIMo 3.1). MRLs and ARfDs valid at the sampling date were used. Figure 5. Time trends in frequency (sample percentage) of MRL and ARfD exceedances. ARfD exceedance based on single-substance point estimate calculations (IESTI in PRIMo 3.1). MRL Figure 5. Time trends in frequency (sample percentage) of MRL and ARfD exceedances. ARfD exceedance based on single-substance point estimate calculations (IESTI in PRIMo 3.1). MRLs and ARfDs valid at the sampling date were used. 4.1.2 Cumulative risk assessments using MCRA The main results from the cumulative risk assessments of Dutch children for the years 2013-2020 is shown in Table 10 and Figure 6. In the table all health effects are listed (if any) which might occur for at least 0.1% of the population. Single-substance assessments, business as usual Two equivalent statistics are shown, the 99.9th percentile of the %ARfD distribution and the probability of critical exposure (POCE), which is the percentage of persondays with exceedance of the ARfD. In addition, the food-substance combinations that are responsible for such exceedances are listed. In the figure each year is represented by the health effect with the highest risk. Due to insufficient availability of amongst others processing factors, these results are provisional exceedances are listed. In the figure each year is represented by the health effect with the highest risk. Due to insufficient availability of amongst others processing factors, these results are provisional. The cumulative assessments indicated that the probability of a critical acute exposure was estimated to be in the range 0.1- 0.4% during the period 2013-2019 (the results for 2020 were affected by an artificial low ARfD value and are therefore not useful to estimate real risk). Nevertheless, the main identified risk drivers were occurrences of chlorpyrifos and chlorpyrifos-methyl in some citrus fruit products such as juices for which no account of processing effects was included in the assessment due to lack of validated data. 23 le 10. Provisional cumulative risk assessment for Dutch children. The ARfD values valid at the of each year were used, including the artificially low values set by Food Compass for oorpyrifos and chloorpyrifos-methyl in 2020. end of each year were used, including the artificially low values set by Food Compass for chloorpyrifos and chloorpyrifos-methyl in 2020. Year Assessment group(s) with potential risk1 Number of active substances / with exposure P99.9 of exposure, as % of ARfD [95% conf. int.] % of persondays exceeding ARfD [95% conf. Single-substance assessments, business as usual int.] food-substance responsible 2013 Developmental 109 / 54 131 [88; 173] 0.22 [0.07; 0.44] Mandarins / chlorpyrifos 0.1% Table grapes / methiocarb 0.1% Nervous 54 / 29 126 [90; 173] 0.20 [0.06; 0.40] Mandarins / chlorpyrifos 0.1% Oranges / chlorpyrifos 0.1% Table grapes / methiocarb 0.1% Eye 39 / 20 106 [69; 148] 0.12 [0.02; 0.24] Mandarins / chlorpyrifos Liver 99 / 47 77 [54; 116] 0.05 [0; 0.16] Table grapes / methiocarb 2014 Developmental 109 / 54 122 [89, 169] 0.19 [0.07; 0.37] Mandarins / chlorpyrifos 0.2% Mandarins / lambda-cyhalothrin 0.1% Nervous 54 / 31 115 [82; 166] 0.16 [0.05; 0.33] Mandarins / chlorpyrifos Eye 39 / 21 106 [77; 148] 0.13 [0.04; 0.27] Mandarins / chlorpyrifos 2015 Developmental 109 / 55 159 [84; 290] 0.33 [0.05; 0.67] Mandarins / chlorpyrifos 0.1% Apples / chlorpyrifos 0.1%, Nervous 54 / 30 158 [84; 290] 0.31 [0.04; 0.63] Mandarins / chlorpyrifos 0.1% Apples / chlorpyrifos 0.1%, Eye 39 / 20 156 [77; 289] 0.28 [0.03; 0.62] Mandarins / chlorpyrifos 0.1% Apples / chlorpyrifos 0.1%, 2016 Developmental 109 / 57 127 [72; 174] 0.18 [0.03; 0.39] Mandarins / chlorpyrifos 0.2%, Apples/ chlorpyrifos 0.1% Nervous 54 / 34 122 [70; 169] 0.17 [0.02; 0.32] Mandarins / chlorpyrifos 0.2%, Apples/ chlorpyrifos 0.1% Eye 39 / 22 118 [67; 167] 0.16 [0.02; 0.31] Mandarins / chlorpyrifos 0.2%, Apples/ chlorpyrifos 0.1% 2017 Developmental 109 / 55 88 [55; 147] 0.07 [0; 0.21] Mandarins / chlorpyrifos Nervous 54 /28 81 [44; 144] 0.06 [0; 0.20 Mandarins / chlorpyrifos Eye 39 / 22 72 [41; 129] 0.05 [0; 0.16] Mandarins / chlorpyrifos 2018 Developmental 109 / 54 67 [49; 103] 0.03 [0; 0.11] 2019 Developmental 109 /54 100 [59; 234] 0.11 [0.01; 0.39] Mandarins / chlorpyrifos Nervous 54 / 30 93 [49; 227] 0.09 [0; 0.35] Mandarins / chlorpyrifos Eye 39 / 22 83 [35; 198] 0.08 [0; 0.30] Mandarins / chlorpyrifos 2020 Nervous 54 / 27 2370 [1100; 3700] 14 [9; 19] Mandarins / chlorpyrifos 4.3% Oranges / chlorpyrifos 2.3% Apples / chlorpyrifos 2.2% Mandarins / chlorpyrifos-methyl 1.8% Oranges / chlorpyrifos-methyl 1.0% Grapefruits / chlorpyrifos 0.2% Grapefruits/ chlorpyrifos-methyl 0.1% Developmental 109 / 52 2350 [1080; 3670] 12 [7; 18] Mandarins / chlorpyrifos 3.6% Oranges / chlorpyrifos 2.1% Mandarins / chlorpyrifos-methyl 1.8% Apples / chlorpyrifos 1.7% Oranges / chlorpyrifos-methyl 0.9% Grapefruits / chlorpyrifos 0.1% Grapefruits/ chlorpyrifos-methyl 0.1% Eye 39 / 18 2030 [940; 3320] 11 [6; 16] Mandarins / chlorpyrifos 5.0% Apples / chlorpyrifos 2.9% Oranges / chlorpyrifos 2.7% Grapefruits / chlorpyrifos 0.3% Adrenal 10 / 8 1360 [395; 2630] 4.9 [3.2; 8.3] Mandarins / chlorpyrifos-methyl 3.7% Oranges / chlorpyrifos-methyl 1.8% Grapefruits/ chlorpyrifos-methyl 0.3% 1 Listed if uncertainty 97.5 % upper limit of cumulative exposure P99.9 is higher than ARfD. 1 Listed if uncertainty 97.5 % upper limit of cumulative exposure P99.9 is higher than ARfD. 2 Median estimate uncertainty not shown 1 Listed if uncertainty 97.5 % upper limit of cumulative exposure P99.9 is higher than ARfD. 2 Median estimate, uncertainty not shown Single-substance assessments, business as usual 2 Median estimate uncertainty not shown 24 Figure 6. Batch comparison analysis in IPGF portal, showing the trend analysis of the index substance ARfD exceedance by the regulatory chosen 99.9th percentile of the cumulative exposure distribution. ARfDs valid at the end of each year were used, including the artificially low values set by Food Compass for chloorpyrifos and chloorpyrifos-methyl in 2020 which explains the artificial high %ARfD value in that year. Bars represent the P0.1-P99.9 variation in exposure on individual-days with the vertical line denoting the median exposure and the upper whisker showing the upper (97.5%) uncertainty bound on the 99.9th percentile. Figure 6. Batch comparison analysis in IPGF portal, showing the trend analysis of the index substance ARfD exceedance by the regulatory chosen 99.9th percentile of the cumulative exposure distribution. ARfDs valid at the end of each year were used, including the artificially low values set by Food Compass for chloorpyrifos and chloorpyrifos-methyl in 2020 which explains the artificial high %ARfD value in that year. Bars represent the P0.1-P99.9 variation in exposure on individual-days with the vertical line denoting the median exposure and the upper whisker showing the upper (97.5%) uncertainty bound on the 99.9th percentile. Figure 6. Batch comparison analysis in IPGF portal, showing the trend analysis of the index substance ARfD exceedance by the regulatory chosen 99.9th percentile of the cumulative exposure distribution. ARfDs valid at the end of each year were used, including the artificially low values set by Food Compass for chloorpyrifos and chloorpyrifos-methyl in 2020 which explains the artificial high %ARfD value in that year. Bars represent the P0.1-P99.9 variation in exposure on individual-days with the vertical line denoting the median exposure and the upper whisker showing the upper (97.5%) uncertainty bound on the 99.9th percentile. Only few food-substance combinations were found that contribute to ARfD exceedance when using the probabilistic MCRA model. In order to have a better view on the food- substance combinations that lead to the highest %ARfD values in the simulations, Table 11 and Table 12 list the main contributing foods, substances and food-substance combinations for the upper 2.5% tail of the cumulative exposure distribution per year for the health effect with the highest %ARfD values. Figure 7, Figure 8 and Figure 9 show the trends in these main contributions graphically. The main risk drivers over th ewhole period appeared to be chlorpyrifos in mandarins, apples and oranges. Single-substance assessments, business as usual Other combinations that were found as occasional risk drivers with more than 10% contribution to the upper exposures were methiocarb in table grapes (in 2013), lambda-cyhalothrin in oranges (in 2019) and chlorpyrifos-methyl in mandarins (in 2020). 25 Table 11 Main foods (left) and substances (right) explaining at least 90% of the 2.5% upper tail of the cumulative exposure distribution of Dutch children for the health effect with the highest risk potential. le 11 Main foods (left) and substances (right) explaining at least 90% of the 2.5% upper tail he cumulative exposure distribution of Dutch children for the health effect with the highest k potential. risk potential. Year Highest contrib. foods % in tail Year Highest contrib. substances % in tail 2013 Mandarins 53 2013 chlorpyrifos 66 Table grapes 20 methiocarb 16 Apples 11 ethephon 8 Oranges 6 lambda-cyhalothrin 5 2014 Mandarins 79 2014 chlorpyrifos 85 Table grapes 8 lambda-cyhalothrin 6 Oranges 5 2015 Apples 61 2015 chlorpyrifos 95 Mandarins 32 2016 Mandarins 57 2016 chlorpyrifos 88 Apples 36 lambda-cyhalothrin 3 2017 Mandarins 78 2017 chlorpyrifos 73 Apples 13 lambda-cyhalothrin 12 imazalil 4 captan 4 2018 Mandarins 67 2018 chlorpyrifos 66 Oranges 19 lambda-cyhalothrin 12 Apples 5 propiconazole 8 ethephon 2 chlorprofam 2 2019 Mandarins 72 2019 chlorpyrifos 69 Oranges 24 lambda-cyhalothrin 19 propiconazole 4 2020 Mandarins 39 2020 chlorpyrifos 72 Oranges 31 chlorpyrifos-methyl 28 Apples 29 26 Figure 7. Trend analysis of the main contributing foods (as percentages) to the upper 2.5% tail of the cumulative exposure distribution. The 5 foods with the highest average contribution over years are shown. Figure 7. Trend analysis of the main contributing foods (as percentages) to the upper 2.5% tail of the cumulative exposure distribution. The 5 foods with the highest average contribution over years are shown. Figure 8. Trend analysis of the main contributing substances (as percentages) to the upper 2.5% tail of the cumulative exposure distribution. The 5 substances with the highest average contribution over years are shown. Figure 8. Trend analysis of the main contributing substances (as percentages) to the upper 2.5% tail of the cumulative exposure distribution. The 5 substances with the highest average contribution over years are shown. 27 Table 12 Main food-substance combinations explaining at least 90% of the 2.5% upper tail of the cumulative exposure distribution of Dutch children for the health effect with the highest risk potential. Single-substance assessments, business as usual Table 12 Main food-substance combinations explaining at least 90% of the 2.5% upper tail of the cumulative exposure distribution of Dutch children for the health effect with the highest risk potential. Year Highest contrib. food+subst % in tail Year Highest contrib. food+subst % in tail 2013 Mandarins / chlorpyrifos 50 2017 Mandarins / chlorpyrifos 68 Table grapes / methiocarb 16 Mandarins / lambda-cyhalothrin 9 Apples / chlorpyrifos 10 Apples / captan 4 Pineapples / ethephon 5 Apples / imazalil 3 Oranges / chlorpyrifos 4 Apples / chlorpyrifos 3 Mandarins / lambda-cyhalothrin 2 Oranges / lambda-cyhalothrin 2 Bananas / ethephon 2 Oranges - chlorpyrifos 2 Oranges / lambda-cyhalothrin 2 2018 Mandarins / chlorpyrifos 54 Table grapes / chlorpyrifos 2 Oranges / chlorpyrifos 9 2014 Mandarins / chlorpyrifos 74 Mandarins / propiconazole 8 Table grapes / chlorpyrifos 4 Oranges / lambda-cyhalothrin 7 Mandarins / lambda-cyhalothrin 4 Mandarins / lambda-cyhalothrin 4 Oranges / chlorpyrifos 4 Potatoes / chlorprofam 2 Apples / chlorpyrifos 3 Table grapes / ethephon 2 Bananas / imazalil 2 Apples / pyraclostrobin 1 2015 Apples / chlorpyrifos 60 Apples / carbendazim 1 Mandarins / chlorpyrifos 38 Apples / acetimiprid 1 2016 Mandarins / chlorpyrifos 53 2019 Mandarins / chlorpyrifos 66 Apples / chlorpyrifos 33 Oranges / lambda-cyhalothrin 15 Mandarins / lambda-cyhalothrin 2 Oranges / chlorpyrifos 3 Oranges / chlorpyrifos 1 Mandarins / propiconazole 3 Oranges / lambda-cyhalothrin 1 Mandarins / lambda-cyhalothrin 2 Table grapes / pyraclostrobin 1 Oranges / dimethoate 2 2020 Oranges / chlorpyrifos 31 Apples / chlorpyrifos 29 Mandarins / chlorpyrifos-methyl 27 Mandarins / chlorpyrifos 12 Figure 9. Trend analysis of the main contributions (as percentages) to the upper 2.5% tail of the cumulative exposure distribution. The 10 food/substance combinations with the highest average contribution over years are shown. Figure 9. Trend analysis of the main contributions (as percentages) to the upper 2.5% tail of the cumulative exposure distribution. The 10 food/substance combinations with the highest average contribution over years are shown. 28 4.2 Example of detailed results (year 2015– Developmental effects) 4.2 Example of detailed results (year 2015– Developmental effects) The whiskers indicate a composed confidence interval, the left whisker is the lower 2.5% limit of p0.1, the right whisker is the upper 97.5% limit of p99.9. Figure 11. Safety chart: bar shows variability of HI (range p0.1 - p99.9) in the population. The whiskers indicate a composed confidence interval, the left whisker is the lower 2.5% limit of p0.1, the right whisker is the upper 97.5% limit of p99.9. Figure 11. Safety chart: bar shows variability of HI (range p0.1 - p99.9) in the population. The whiskers indicate a composed confidence interval, the left whisker is the lower 2.5% limit of p0.1, the right whisker is the upper 97.5% limit of p99.9. Table 13. Risks for Dutch children based on 2015 data (developmental effects). HI = Hazard Index. POCE = Probability of Critical Exposure. p = percentile. Unc = Uncertainty. HI (p0.1) HI (p50) HI (p99.9) HI (p99.9) - Unc (p97.5) POCE (%) POCE (%) lower bound (p2.5) POCE (%) upper bound (p97.5) 0.0003 0.043 1.6 2.9 0.33 0.05 0.67 Zooming in on the individual substance contributions (Figure 12, Figure 13), by far the largest contribution to the cumulative risk is seen to come from Chlorpyrifos. Table 13. Risks for Dutch children based on 2015 data (developmental effects). HI = Hazard Index. POCE = Probability of Critical Exposure. p = percentile. Unc = Uncertainty. HI (p0.1) HI (p50) HI (p99.9) HI (p99.9) - Unc (p97.5) POCE (%) POCE (%) lower bound (p2.5) POCE (%) upper bound (p97.5) 0.0003 0.043 1.6 2.9 0.33 0.05 0.67 Table 13. Risks for Dutch children based on 2015 data (developmental effects). HI = Hazard Index. POCE = Probability of Critical Exposure. p = percentile. Unc = Uncertainty. Table 13. Risks for Dutch children based on 2015 data (developmental effects). HI = Hazard Index. POCE = Probability of Critical Exposure. p = percentile. Unc = Uncertainty. HI (p0.1) HI (p50) HI (p99.9) HI (p99.9) - Unc (p97.5) POCE (%) POCE (%) lower bound (p2.5) POCE (%) upper bound (p97.5) 0.0003 0.043 1.6 2.9 0.33 0.05 0.67 Zooming in on the individual substance contributions (Figure 12, Figure 13), by far the largest contribution to the cumulative risk is seen to come from Chlorpyrifos. Zooming in on the individual substance contributions (Figure 12, Figure 13), by far the largest contribution to the cumulative risk is seen to come from Chlorpyrifos. Figure 12. 4.2 Example of detailed results (year 2015– Developmental effects) In this section detailed results are shown for the batch analysis of the year 2015, which was the year with the highest observed risk index (excluding 2020, for which the high risk was due to an artifical low ARfD). These results and similar results for other years and health effects are available for users of the IPGF portal. As in most years in the trend analysis, developmental effects were identified as the health effects of primary concern. The assessment group for developmental effects consists of 110 active substances, but one of these (fenoxaprop-P-ethyl) excluded from the assessment because of a missing ARfD value. For cumulative assessments, oxamyl was selected as the index substance, which means that all exposures are expressed as oxamyl equivalents based on ARfD ratios used as relative potency factors. For 54 of the remaining 109 active substances no positive exposure from the diet was found. Therefore, the assessment group cumulated the risks from effectively 55 substances that were found in the diet of the Dutch children. In the simulations, at least one of these substances was present in the diet every day (100% exposure). The cumulative exposure (in oxamyl equivalents) divided by the ARfD of oxamyl (which happens to be 1 µg/kg/day) specifies a hazard index (HI) distribution (Figure 10, Figure 11). EC, EFSA, RIVM and NVWA have agreed on using a 99.9% level of protection. Therefore, the cumulative exposure and HI distributions are evaluated at the 99.9th percent point (Table 13). Whereas the median estimate of HI is well below 1, the estimate of P99.9 is 1.6 with an uncertainty upper bound of 2.9. This means that for 0.1% of the children the cumulative exposure is estimated to be 1.6 times the ARfD and could be up to 2.9 times the ARfD. Another way to express these same results is to state that the probability of a critical exposure (POCE) is estimated as 0.33% with an uncertainty upper bound of 0.67%. Figure 10. Hazard index distribution for Dutch children 2015, developmental effects, estimated from 10,000 Monte Carlo iterations. Figure 10. Hazard index distribution for Dutch children 2015, developmental effects, estimated from 10,000 Monte Carlo iterations. 29 Figure 11. Safety chart: bar shows variability of HI (range p0.1 - p99.9) in the population. 4.2 Example of detailed results (year 2015– Developmental effects) Contribution to total (left) and upper 0.1% tail (right) cumulative exposure from individual substances. Figure 12. Contribution to total (left) and upper 0.1% tail (right) cumulative exposure from individual substances. 30 dex (CUMULATIVE) and hazard quotients f Figure 13. Hazard index (CUMULATIVE) and hazard quotients for contributing substances. 31 Zooming in on the individual vegetable and fruit contributions (Figure 14), the largest contributions to the cumulative risk is seen to come from Apples, Mandarins and Oranges. In terms of the consumed products, these are identified to be mainly the juiced products, i.e. apple juice, mandarin juice and orange juice. Zooming in on the individual vegetable and fruit contributions (Figure 14), the largest contributions to the cumulative risk is seen to come from Apples, Mandarins and Oranges. In terms of the consumed products, these are identified to be mainly the juiced products, i.e. apple juice, mandarin juice and orange juice. Zooming in on the individual vegetable and fruit contributions (Figure 14), the largest contributions to the cumulative risk is seen to come from Apples, Mandarins and Oranges. In terms of the consumed products, these are identified to be mainly the juiced products, i.e. apple juice, mandarin juice and orange juice. Figure 14. Contribution from individual vegetable and fruit products as measured to total (upper left) and upper 0.1% tail (upper right) cumulative exposure and contribution from vegetable and fruit products as consumed to total (lower left) and upper 0.1% tail (lower right) cumulative exposure. Figure 14. Contribution from individual vegetable and fruit products as measured to total (upper left) and upper 0.1% tail (upper right) cumulative exposure and contribution from vegetable and fruit products as consumed to total (lower left) and upper 0.1% tail (lower right) cumulative exposure. If we consider the most detailed level, we see that indeed Chlorpyrifos in apple, mandarin and orange juices is primarily responsible for the higher exposures (Figure 15. Contribution from combinations of substance and vegetable and fruit product as consumed to total (left) and upper 0.1% tail (right) cumulative exposure.). Figure 15. Contribution from combinations of substance and vegetable and fruit product as consumed to total (left) and upper 0.1% tail (right) cumulative exposure. Figure 15. Contribution from combinations of substance and vegetable and fruit product as consumed to total (left) and upper 0.1% tail (right) cumulative exposure. Discussion and conclusions 5 The IPGF portal has been made available for Food Compass as primary responsible stakeholder for food safety of vegetables and fruits in the Netherlands to perform cumulative acute risk assessments. Cumulative assessments, in contrast to IESTI single-substance assessments, address concerns of the EC and the general public about potential mixture effects. Probabilistic assessments are more realistic than simple conservative calculations (IESTI). It was shown that cumulative assessment provides a useful addition for Food Compass or other private stakeholders to assess the combined risk of multiple chemicals. In this report, batch analyses per year were performed for investigating the trends in the period 2013-2020. The results presented in this report provide insight in the trends over the years and the high contributing food products and substances driving the (mixture) risk. It is essential to note that the data are not perfect and there are many aspects where a lack of data (quality) is identified, which may lead to a potential bias in the calculations. For a part, this lack of data can be addressed by collecting more data and resolving the quality issues. For complex modelling this seems to be normal, and therefore we consider optimisation of data organisation as a form of a Retain & Refine (R&R) strategy (Kennedy et al. 2020). If a risk assessment with conservative elements due to imperfect data organisation shows no risk, it is not needed to improve the data and models (Retain). However, if potential risks are identified, refinement of the data and use of advanced models for analysis may be indicated (Refine). The results of the current analysis highlight chlorpyrifos as the main risk driver. However, Chlorpyrifos (as well as chlorpyrifos-methyl) is now considered to be mutagenic genotoxic and is no longer allowed in the European Union. For these substances an ARfD is no longer available as it is considered mutagenic. This generates a problem for cumulative assessments which depend on estimation of the relative potencies. In this study we used ratios of ARfDs as relative potency factors. In this report, we therefore kept using ARfD values, in the form as were made available by GroentenFruit Huis. For Chlorpyrifos and Chlorpyrifos-methyl this led to an artificial high-risk estimate for the year 2020 because GroentenFruit Huis has set the corresponding ARfDs to artificial low values (0.0001 mg/kg bw/d) per 13-11-2020, just to generate alerts. 4.2 Example of detailed results (year 2015– Developmental effects) 32 A further drill-down (Table 14) reveals that chlorpyrifos in orange juice has a processing factor of 0.025, i.e. the concentrations are assumed to be 40 times lower in orange juice as compared to the raw agricultural product oranges. However, it is also seen that no processing factors were used for apple juice, mandarin juice and for a different form of orange juice (Juicing, Concentration/evaporation). This suggests that exposure from these sources is over-estimated. Table 14. Processing factors for risk drivers. Substan ce name Substan ce code Food name Food code Processing type Contribution (%) mean Processing factor chlorpyrifos 2921882 Apples P0130010A Juicing 58.31211028 1 chlorpyrifos 2921882 Apples P0130010A Unspecified 1.043602482 1 chlorpyrifos 2921882 Mandarins P0110050A Juicing 30.42689077 1 chlorpyrifos 2921882 Mandarins P0110050A Juicing, Concentration / evaporation 0.091850867 1 chlorpyrifos 2921882 Oranges P0110020A Juicing, Concentration / evaporation 1.428885321 1 chlorpyrifos 2921882 Oranges P0110020A Juicing 0.046311439 0.025 Table 14. Processing factors for risk drivers. 33 Discussion and conclusions As a consequence, the results of the current trend analysis show an increase in perceived risk in 2020 (note that the ARfD valid at 31-12-2020 was used for all exposures in the batch). It should be noted that this increase indicates a higher frequency of alerts only and not an increase in real risk (as someone might conclude from the 2020 results in chapter 4). For a more realistic comparison between years, it might be useful to perform calculations with the same ARfD values across the years. Due to a missing ARfD value, fenoxaprop-P-ethyl was not included in the cumulative calculations. It could be considered to use the ARfD of fenoxaprop-P instead. In this case study we grouped the pesticides in 15 CAGs corresponding with level 1 (organ level) CAGs as in Nielsen et al. (2012). In this way we covered a variety of potential health effects to illustrate the application of the proposed methodology. However, it should be remarked that these CAGs were not specifically derived for acute risk assessment, but rather for chronic risk assessment, i.e. following cumulative exposure over time. Specific CAGs for acute health risks have only been derived by EFSA for two specific neurological effects at level 2 (EFSA 2019c, van Klaveren et al. 2019a). The process to define more CAGs is ongoing. Following the approach of te Biesebeek et al. (2021), the hazard characterisations used in the cumulative exposure assessments were ARfDs applying to the so-called critical effect. For some CAGs, the ARfD of a substance may not relate to the specific organ of the CAG, but to an adverse effect on another organ. The use of the ARfD may 34 therefore be seen as a conservative estimate of an organ-specific hazard dose and may lead to an overestimation of the risk. therefore be seen as a conservative estimate of an organ-specific hazard dose and may lead to an overestimation of the risk. For refinement of the calculations performed in this study we identify the following types of data that were not yet optimal: For refinement of the calculations performed in this study we identify the following types of data that were not yet optimal: 1) Laboratory scopes did not always match with the reported substance measurements, and there is large number of samples and measurement reports with inconsistencies. 2) The set of processing factors was incomplete, as illustrated for example in Table 14. Discussion and conclusions This can lead to an overestimation of the exposure, which was also observed in van Klaveren et al. (2019a). 3) In this report, we assumed that our collection of ARfD values represented the valid ARfD values during the period 2013-2020. However, it can be doubted if this collection is complete. The current GroentenFruit Huis web service only provides the most recent ARfD value. It should be discussed how the collection of historical ARfD values can be completed, and in fact, if this is considered a necessary approach. 4) Use of ARfD values to characterise hazard is a conservative approach because in a group of substances the ARfD is related to the the most critical health effect for each substance individually. If specific hazard characterisation data for each assessment group are available for all substaces in the group, these could be used (te Biesebeek et al. 2021). The IPGF portal will be further developed in 2022, in line with developments in the methodology of mixture risk assessments by RIVM and EFSA. An update of the trend analysis in this report will be reported. Also, in this study we focused on cumulative risk assessments of year batches using the new IPGF portal and MCRA, in comparison to single-sample analyses for MRL and/or ARfD exceedances. In addition to the analyses described in this report, several other approaches are possible, some of them already implemented and other to be discussed: • Instead of the Monte Carlo simulations, it would also be possible to perform probabilistic calculations for each single sample separately against a background of other samples, and then to summarise the sample results per year. In fact, this was originally the intention, but the computational load turned out to be very high. Further discussion might be needed if this would be a useful addition to the current approach. • Here we have focused on trend analysis, i.e. a retrospective assessment. Another potential use of the IPGF portal would be a real-time use, where cumulative risk assessments for single samples representing product consignments could be used to decide on the acceptability of these consignments. • In the ongoing discussions, retailers have set additional stringent criteria. 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Database of processing techniques and processing factors compatible with the EFSA food classification and description system FoodEx 2. Objective 3: European database of processing factors for pesticides in food. EFSA supporting publication 2018: 15( 11):EN-1510. 50 pp. https://doi.org/10.2903/sp.efsa.2018.EN-1510 van der Voet H, Kruisselbrink JW, de Boer WJ, van Lenthe MS, van den Heuvel JJB, Crépet A, Kennedy MC, Zilliacus J, Beronius A, Tebby C, Brochot C, Luckert C, Lampen A, Rorije E, Sprong C and van Klaveren JD, 2020. The MCRA toolbox of models and data to support chemical mixture risk assessment. Food and Chemical Toxicology, 138, 111185. https://doi.org/10.1016/j.fct.2020.111185 van Klaveren J, Kruisselbrink JW, de Boer WJ, van Donkersgoed G, te Biesebeek JD, Sam M and van der Voet H, 2019a. Cumulative dietary exposure assessment of pesticides that have acute effects on the nervous system using MCRA software. EFSA supporting publication 2019:EN-1708 https://doi.org/10.2903/sp.efsa.2019.en-1708 van Klaveren JD, Kruisselbrink JW, de Boer WJ, van Donkersgoed G, te Biesebeek JD, Sam M and van der Voet H, 2019b. Cumulative dietary exposure assessment of pesticides that have chronic effects on the thyroid using MCRA software. EFSA supporting publication 2019:EN-1707. https://doi.org/10.2903/sp.efsa.2019.EN-1707. 36 Supplementary material Available at https://library.wur.nl/WebQuery/doi/562203 File Description Design and implementation of the IPGF Portal (v2.0.0-beta.1) Document describing technical design and implementation of the IPGF portal. EUProcessingFactorsDB_MCRA_FCCAS.1.0.3.zip Processing factors dataset (in MCRA format) used in cumulative exposure assessments. DTUCAG.1.2.0 Cumulative assessment groups dataset (in MCRA format) used in cumulative exposure assessments. Substances.csv Snapshot of substances catalogue (incl. p 1532 PARAM mapping and hierarchy) at the time of calculating the results. LabScopes.zip Zip file containing three csv files describing the laboratory scopes user for import of the concentration data used in the calculations. ARfDs.csv Snapshot of the ARfDs catalogue as used in the calculations. Supplementary material Available at https://library.wur.nl/WebQuery/doi/562203 Supplementary material Correspondentie adres voor dit rapport: Postbus 16 6700 AA Wageningen T 0317 48 40 85 www.wur.nl/ Openbaar Rapport De missie van Wageningen University & Research is ‘To explore the potential of nature to improve the quality of life’. Binnen Wageningen University & Research bundelen Wageningen University en gespecialiseerde onderzoeksinstituten van Stichtin g Wageningen Research hun krachten om bij te dragen aan de oplossing van belangrijke vragen in het domein van gezonde voeding en leefomgevin g. Met ongeveer 30 vestigingen, 5.000 medewerkers en 10.000 studenten behoort Wagening en University & Research wereldwijd tot de aanspreken de kennisinstellin gen binnen haar domein. De integrale benadering van de vraagstukken en de samenw erking tussen verschillend e disciplines vormen het hart van de unieke Wageningen aanpak. 37

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